TOPO TA Manual

User Manual

NuPAGE

Technical Guide

General information and protocols for using the

NuPAGE

electrophoresis system

Rev. date: 29 October 2010

Manual part no. IM-1001

MAN0003188

Contents

NuPAGE

Precast Gels.................................................................................................5

General Information.......................................................................................................................................5

Description of the NuPAGE

Electrophoresis System ..............................................................................6

NuPAGE

Gel Specifications ........................................................................................................................8

Gel Selection ....................................................................................................................................................9

Well Volume..................................................................................................................................................10

Gel Staining ...................................................................................................................................................11

Methods .......................................................................................................................12

General Guidelines for Samples and Buffers............................................................................................12

Preparing Buffers for Denaturing Electrophoresis ..................................................................................14

Preparing Buffers for Non-Denaturing Electrophoresis .........................................................................16

Electrophoresis of NuPAGE

Gels .............................................................................................................17

Opening Novex

Pre-Cast Gel Cassettes...................................................................................................19

Silver Staining ...............................................................................................................................................20

Coomassie Staining ......................................................................................................................................24

SYPRO

Ruby Staining ................................................................................................................................28

Gel Drying .....................................................................................................................................................31

Western Blotting ...........................................................................................................................................34

Using ZOOM

Gels ......................................................................................................................................40

Calibrating Protein Molecular Weight.......................................................................................................42

Troubleshooting............................................................................................................................................45

Appendix......................................................................................................................48

Accessory Products ......................................................................................................................................48

Recipes............................................................................................................................................................50

Gel Migration Chart .....................................................................................................................................54

Gel Conversion Chart...................................................................................................................................55

Technical Support.........................................................................................................................................56

Purchaser Notification .................................................................................................................................58

References ......................................................................................................................................................59

NuPAGE

Precast Gels

General Information

Purpose of the

Guide

The NuPAGE

Technical Guide contains information about the NuPAGE

Electrophoresis System and is intended to supplement the NuPAGE

Bis-Tris

Gel Instruction Card (IM-8042) and the NuPAGE

Tris-Acetate Gel Instruction

Card (IM-1025). Complete protocols for sample preparation, buffer preparation,

electrophoresis, staining, and blotting are provided in this guide.

For additional information, contact Technical Support (see page 56) or download

the manuals from our website at www.invitrogen.com.

For description of the NuPAGE

electrophoresis system, see page 6–7.

Storage and Shelf

life

Store NuPAGE

Novex

Bis-Tris Gels at 4–25C and NuPAGE

Novex

Tris-

Acetate Gels at +4

C.

The NuPAGE

Novex

Bis-Tris Gels have a shelf life of 12 months when stored

at 4

–25C.

The NuPAGE

Novex

Tris-Acetate Gels have a shelf life of 8 months when

stored at 4

C.

Do not freeze NuPAGE

Gels.

Using expired gels or improperly stored gels may result in poor band

resolution.

Packaging

The NuPAGE

Pre-Cast Gels are individually packaged in clear pouches with

10 mL of Packaging Buffer.

Handling the Gels

The Packaging Buffer contains low levels of residual acrylamide monomer and

0.02% sodium azide. Gloves should be worn at all time when handling gels.

Warning: This product contains a chemical (acrylamide) known to the state of

California to cause cancer. To obtain a SDS, see page 56.

Intended Use

For research use only. Not intended for human or animal diagnostic or

therapeutic uses.

Description of the NuPAGE

Electrophoresis System

Introduction

The NuPAGE

Bis-Tris Electrophoresis System is a revolutionary neutral pH,

pre-cast, discontinuous SDS-PAGE mini-gel system providing maximum

stability of both proteins and gel matrix during electrophoresis, and better band

resolution than other gel systems.

The most widely used gel system for separating a broad range of proteins by

SDS-PAGE is the Laemmli system (Laemmli, 1970). The highly alkaline

operating pH of the Laemmli system may cause band distortion, loss of

resolution, or artifact bands. The major causes of poor band resolution with the

Laemmli system are:

 Hydrolysis of polyacrylamide at the high gel casting pH of 8.7 resulting in a

short shelf life of 4–6 weeks

 Chemical modifications such as deamination and alkylation of proteins due

to the high pH (9.5) of the separating gel

 Reoxidation of reduced disulfides from cysteine containing proteins as the

redox state of the gel is not constant

 Cleavage of Asp-Pro bond of the proteins when heated at 100C in the

Laemmli sample buffer, pH 5.2 (Kubo, 1995).

Advantages of the

NuPAGE

Electrophoresis

System

The neutral operating pH (pH 7.0) of the NuPAGE

Gels and buffers provide

following advantages over the Laemmli system:

 Longer shelf life of 8–12 months due to improved gel stability (see page 5)

 Im

proved protein stability during electrophoresis at neutral pH resulting in

sharper band resolution and accurate results (Moos et al, 1998)

 Complete reduction of disulfides under mild heating conditions (70C for

10 minutes) and absence of cleavage of Asp-Pro bonds using the NuPAGE

LDS Sample buffer (pH >7.0 at 70C)

 Reduced state of the proteins maintained during electrophoresis and

blotting of the proteins when using the NuPAGE

Antioxidant

NuPAGE

Electrophoresis

System

Components

The NuPAGE

Electrophoresis System consists of:

 NuPAGE

Novex

Bis-Tris [Bis (2-hydroxyethyl) imino-tris

(hydroxymethyl) methane-HCl] Pre-Cast Gels for separating small to mid-

size molecular weight proteins

 NuPAGE

Novex

Tris-Acetate Pre-Cast Gels for separating large

molecular weight proteins

 NuPAGE

LDS (Lithium dodecyl sulfate) Sample Buffer

 NuPAGE

Reducing Agent

 NuPAGE

Antioxidant

 NuPAGE

MES [2-(N-morpholino) ethane sulfonic acid] SDS or MOPS [3-

(N-morpholino) propane sulfonic acid] SDS Running Buffer for NuPAGE

Novex

Bis-Tris Gels

 NuPAGE

Tris-Acetate SDS Running Buffer for NuPAGE

Novex

Tris-

Acetate Gels

 NuPAGE

Transfer Buffer for blotting of NuPAGE

Novex

Pre-Cast Gels

Continued on next page

Description of the NuPAGE

Electrophoresis System, Continued

NuPAGE

Bis-Tris

Discontinuous

Buffer System

The NuPAGE

Bis-Tris discontinuous buffer system involves three ions:

 Chloride (



) from the gel buffer and serves as a leading ion due to its high

affinity to the anode relative to other anions in the system. The gel buffer

ions are Bis-Tris

and Cl



(pH 6.4).

 MES or MOPS (



) serve as the trailing ion in the running buffer. The running

buffer ions are Tris

, MOPS



/MES



, and dodecylsulfate



(pH 7.3–7.7).

 Bis-Tris (

) is the common ion present in the gel buffer and running buffer.

The combination of the lower pH gel buffer (pH 6.4) and the running buffer

(pH 7.3–7.7) results in a significantly lower operating pH of 7 during

electrophoresis.

NuPAGE

Tris-

Acetate

Discontinuous

Buffer System

The NuPAGE

Tris-Acetate discontinuous buffer system involves three ions:

 Acetate (



) from the gel buffer and serves as a leading ion due to its high

affinity to the anode relative to other anions in the system. The gel buffer

ions are Tris

and Acetate



(pH 7.0).

 Tricine (



) from the running buffer serves as the trailing ion. The running

buffer ions are Tris

, Tricine



, and dodecylsulfate



(pH 8.3).

 Tris (

) is the common ion present in the gel buffer and running buffer. The

Tris-Acetate system also operates at a significantly lower operating pH of

8.1 during electrophoresis.

Separation Range

of Proteins

The NuPAGE

Gels have a wider range of separation on a single gel and also

separate proteins evenly through the low and high molecular weight ranges

compared to existing gels. Due to these advantages, most proteins are well

resolved on one of the five NuPAGE

gels (see Applications, page 9).

By combining any of the NuPAGE

Novex

Bis-Tris Gels with the MES SDS or

MOPS SDS Running Buffer, you can obtain six separation ranges for resolving

proteins over a wide molecular weight range of 1–200 kDa. The NuPAGE

Novex

Tris-Acetate gels resolve proteins in the molecular weight range of

36–400 kDa.

NuPAGE

Gel Specifications

Introduction

The NuPAGE

Novex

Pre-Cast Gel cassette is 10 cm × 10 cm in size, and

designed for use with the XCell SureLock

™

Mini-Cell and XCell6

™

MultiGel Unit

(see page 48 for ordering information).

NuPAGE

Novex

Pre-Cast Bis-Tris Gels are available for resolving proteins in

the range of 1–200 kDa, and NuPAGE

Novex

Pre-Cast Tris-Acetate Gels

resolve proteins in the range of 36–400 kDa (depending upon the acrylamide

percentage of the gel and buffer system being used). Refer to the Novex

Gel

Migration Charts (page 54) to find the gel with the region of maximum

resolution best suited for your sample.

Specifications

Gel Matrix: Acrylamide/Bisacrylamide

Gel Thickness: 1.0 mm

Gel Size: 8 cm × 8 cm

Cassette Size: 10 cm × 10 cm

Cassette Material: Styrene Copolymer (recycle code 7)

Sample Well Configuration: 1, 9, 10, 12, 15, 17-well, 2D-well, and IPG well

NuPAGE

Gel

Formulations

The NuPAGE

Novex

Pre-Cast Gels are available in different acrylamide

concentrations (see the table below).

The NuPAGE

Gels do not contain SDS. However, they are designed for

performing denaturing gel electrophoresis (see Applications, next page).

Gel Type Formulation Stacking Gel Separating Gel pH

NuPAGE

Novex

Bis-Tris Gels

Bis-Tris-HCl buffer (pH 6.4),

Acrylamide, Bis-acrylamide,

APS, Ultrapure water

4% 10%, 12%, 4–12% 7.0

NuPAGE

Novex

Tris-Acetate Gels

Tris-base, Acetic acid,

Acrylamide, Bis-acrylamide,

TEMED, APS, Ultrapure water

3.2% 7%, 3–8% 8.1

Crosslinker

The crosslinker concentration for the NuPAGE

Novex

Pre-Cast Gel ranges

from 3.8–5% depending on the region of the gel.

Gel Selection

Choosing a

NuPAGE

Gel for

Your Application

To obtain the best results, it is important to choose the correct gel percentage,

buffer system, gel format, and thickness for your application. NuPAGE

Pre-

Cast Gels are compatible with protein sequencing using Edman sequencing

from the gel, or from PVDF membranes.

Review Applications (below), and Well Volume (page 10) to determine the

type of gel that is best suited for your application.

efer to the NuPAGE

Gel Migration Chart (page 54) to find the gel with the

region of maximum resolution best suited for your sample. The leading protein

molecules should migrate about 70% of the length of gel for best resolution.

Applications

Separation of proteins over a wide range of molecular weights

Use NuPAGE

Bis-Tris Gels with NuPAGE

MOPS SDS Running Buffer to

resolve proteins (14–200 kDa) under denaturing conditions.

Separation of low molecular weight proteins

Use NuPAGE

Bis-Tris Gels with NuPAGE

MES SDS Running Buffer Buffer to

resolve small molecular weight proteins (2–200 kDa) under denaturing

conditions.

Separation of high molecular weight proteins

Use NuPAGE

Tris-Acetate Gels with NuPAGE

Tris-Acetate SDS Running

Buffer to resolve high molecular weight proteins (36–400 kDa) under

denaturing conditions, or with Novex

Tris-Glycine Native Running Buffer to

resolve high molecular weight proteins under non-denaturing (native)

conditions.

Note: Do not use the NuPAGE

Bis-Tris Gels with NuPAGE

MOPS or MES

Running Buffer without SDS for native gel electrophoresis. This buffer system

may generate excessive heat resulting in poor band resolution. The protein of

interest may not migrate very well in a neutral pH environment if it is not

charged.

2D separation of proteins

The ZOOM

Gels are specifically designed for second dimension

electrophoresis of 7.0 cm IPG strips.

Well Volume

Recommended

Loading Volumes

The recommended loading volumes and protein load per band by the detection

method are provided in the table below.

Note: The 9- and 17-wells are compatible with any eight-channel pipette used

for loading samples from 96-well plates. An additional lane is included for

loading protein molecular weight standard.

Maximum Protein Load Per Band by Detection Method Well Types Maximum Load

Volume

Coomassie Staining Silver Staining Immunoblotting

1 well

1.0 mm

700 μL 12 μg/band

2D well

1.0 mm

1.5 mm

400 μL

600 μL

12 μg/band

IPG well

1.0 mm

7 cm IPG Strip N/A

9 well

1.0 mm

28 μL 0.5 μg/band

10 well

1.0 mm

1.5 mm

25 μL

37 μL

0.5 μg/band

12 well

1.0 mm

20 μL 0.5 μg/band

15 well

1.0 mm

1.5 mm

15 μL

25 μL

0.5 μg/band

17 well

1.0 mm 15 μL 0.5 μg/band

Scale your sample

load for the

sensitivity of your

silver staining kit.

For use with the

SilverQuest

™

SilverXpress

Silver

Staining Kits, we

recommend a

protein load of

1 ng/band.

Scale your

sample load

according to the

sensitivity of

your detection

method.

Choosing the

Appropriate Well

for Your

Application

Choose the type of well for your application based upon the volume of your

sample. The more wells a comb has, and the thinner the gel is, the lower the

sample loading volume.

Note: Proteins transfer out of a 1.0 mm gel more easily than from a 1.5 mm gel.

Gel Staining

Staining NuPAGE

Gels

The NuPAGE

Novex

Pre-Cast Gels are compatible with most silver staining

protocols. We recommend using the SilverQuest

™

Silver Staining Kit or the

SilverXpress

Silver Staining Kit (see pages 20–23) for silver staining of

NuPAGE

Gels.

The NuPAGE

Novex

Pre-Cast Gels are compatible with any of the standard

Coomassie staining procedures. The protocols that are accelerated by heat are

preferable as the heat serves as a “fix” for proteins, especially smaller peptides.

The SimplyBlue

™

SafeStain and Novex

Colloidal Coomassie Blue Staining Kit

(see pages 24–27) are recommended for staining NuPAGE

Gels.

The NuPAGE

Novex

Pre-Cast Gels are also compatible with copper or zinc

staining, and fluorescent stains like the SYPRO

Ruby gel stain (see pages 28–

30)

Methods

General Guidelines for Samples and Buffers

Introduction

General information on the sample buffer and reducing agent is provided

below. For sample and buffer preparation protocols, see page 14. Instructions

for preparing running buffers for denaturing and non-denaturing

electrophoresis are provided on page 50–52.

Recommended

Buffers

The recommended running buffer and sample buffer for each NuPAGE

Novex

Pre-Cast Gel is listed in the table below. Prepare your sample in the appropriate

sample buffer so that the final concentration of the sample buffer is 1X. Running

buffer must be diluted to 1X final concentration before use.

See page 48 for ordering information on pre-mixed buffers. See pages 50–52 for

recipes if you are preparing your own buffers.

Gel Type Running Buffer Sample Buffer

NuPAGE

Novex

Bis-Tris

Gel (SDS-PAGE)

NuPAGE

MES or MOPS SDS

Running Buffer

NuPAGE

LDS Sample Buffer

NuPAGE

Novex

Tris-

Acetate Gels (SDS-PAGE)

NuPAGE

Tris-Acetate Running

Buffer

NuPAGE

LDS Sample Buffer

NuPAGE

Novex

Tris-

Acetate Gels (Native-PAGE)

Novex

Tris-Glycine Native

Running Buffer

Novex

Tris-Glycine Native Sample

Buffer

NuPAGE

LDS

Sample Buffer

Use the NuPAGE

LDS Sample Buffer for preparing samples when performing

denaturing gel electrophoresis with NuPAGE

Gels. The slightly alkaline pH of

the NuPAGE

LDS Sample Buffer (pH 8.4) provides the optimal conditions for

reduction of protein disulfide bonds, and denaturation.

The NuPAGE

LDS Sample Buffer is a 4X concentrated solution containing

twice as much dodecylsulfate as the 2X concentration of Novex

Tris-Glycine

SDS or Tricine SDS Sample Buffer. The buffer also contains more glycerol,

resulting in increased viscosity.

Bring the NuPAGE

LDS Sample Buffer to room temperature (25°C) before use

to make pipetting the buffer easier.

Tracking Dye

The NuPAGE

LDS Sample Buffer uses Coomassie G250 and Phenol Red as

tracking dyes instead of bromophenol blue. Coomassie G250 gives a sharp dye

front with both MES and MOPS SDS Running Buffers and migrates much

closer to the moving ion front than bromophenol blue. Bromophenol blue runs

more slowly than some peptides with the MES SDS Running Buffer. This

ensures that small peptides do not run off the gel.

The concentration of the tracking dye (Coomassie G250) is increased in the

NuPAGE

LDS Sample Buffer to enhance viewing of the dye front.

Continued on next page

General Guidelines for Samples and Buffers, Continued

Reducing Agent

Use the NuPAGE

Reducing Agent to prepare samples for reducing gel

electrophoresis. The NuPAGE

Reducing Agent (10X) contains 500 mM

dithiothreitol (DTT) and is available in a ready-to-use, stabilized liquid form.

As an alternative, -mercaptoethanol can be used with NuPAGE

Gels at a final

concentration of 2.5%.

Add the reducing agent to the sample up to an hour before loading the gel.

Avoid storing reduced samples for long periods, even if they are frozen.

Reoxidation of samples occur during storage and produce inconsistent results.

NuPAGE

Antioxidant

Use the proprietary NuPAGE

Antioxidant in the running buffer of the Upper

(cathode) Buffer Chamber when performing electrophoresis under reducing

conditions to prevent sample reoxidation and maintain the proteins in a reduced

state. DTT and -mercaptoethanol tend to remain at the top of the gel, and do

not co-migrate with the sample in the neutral pH environment of NuPAGE

Gels. Disulfide bonds are less reactive at neutral pH and less likely to reoxidize

than in higher pH systems, but some reoxidization may occur during

electrophoresis in the absence of an antioxidant, and cause band diffusion.

The NuPAGE

Antioxidant migrates with the proteins during electrophoresis,

and protects disulfide bonds and sensitive amino acids (e.g., methionine and

tryptophan) from oxidizing.

The NuPAGE

Antioxidant is NOT compatible with gel systems other than the

NuPAGE

system because the antioxidant is not efficient at the higher pHs of

other gel systems. For best results, use the NuPAGE

Antioxidant with reduced

and alkylated samples.

Important

Do not use the NuPAGE

Antioxidant as a sample reducing agent. The

antioxidant is not efficient in reducing disulfide bonds on its own, and using it to

reduce samples results in substantial background smearing in the lane due to

partially reduced bands.

NuPAGE

SDS

Running Buffer

Three types of NuPAGE

Running Buffers are available for denaturing

electrophoresis:

 NuPAGE

MES SDS Running Buffer is used with NuPAGE

Novex

Bis-Tris

Gels to resolve small molecular weight proteins

 NuPAGE

MOPS SDS Running Buffer is used with NuPAGE

Novex

Bis-

Tris Gels to resolve mid-size proteins

 NuPAGE

Tris-Acetate SDS Running Buffer is used with NuPAGE

Novex

Tris-Acetate Gels to resolve high molecular weight proteins

MES has a lower pKa than MOPS, making the NuPAGE

MES SDS Running

Buffer faster than the NuPAGE

MOPS SDS Running Buffers. The difference in

ion migration affects stacking and results in a difference in protein separation

range between these buffers.

For native gel electrophoresis with NuPAGE

Novex

Tris-Acetate Gels, use

the Novex

Tris-Glycine Native Running Buffer.

Preparing Buffers for Denaturing Electrophoresis

Materials Supplied

by the User

The following reagents are needed to prepare samples for denaturing

electrophoresis. Ordering information for pre-mixed buffers is on page 48. If

you are preparing your own buffers, recipes are provided on page 50–52.

 Protein sample

 Deionized water

For Sample Preparation

 NuPAGE

LDS Sample Buffer

 NuPAGE

Reducing Agent

For Running Buffer Preparation

 NuPAGE

MES SDS Running Buffer (for small proteins on NuPAGE

Bis-

Tris Gels)

 NuPAGE

MOPS SDS Running Buffer (for mid-sized proteins NuPAGE

Bis-Tris Gels)

 NuPAGE

Tris-Acetate Running Buffer (for NuPAGE

Tris-Acetate Gels)

 NuPAGE

Antioxidant

Preparing

Samples

Instructions are provided below to prepare reduced or non-reduced samples

for denaturing gel electrophoresis using the NuPAGE

Novex

Bis-Tris or Tris-

Acetate Gels.

For reduced sample, add the reducing agent immediately prior to

electrophoresis to obtain the best results.

Reagent Reduced Sample Non-reduced Sample

Sample x μL x μL

NuPAGE

LDS Sample Buffer (4X) 2.5 μL 2.5 μL

NuPAGE

Reducing Agent (10X) 1 μL —

Deionized Water to 6.5 μL to 7.5 μL

Total Volume 10 μL 10 μL

Running Reduced

and Non-Reduced

Samples

For optimal results, we do not recommend running reduced and non-reduced

samples on the same gel.

If you do choose to run reduced and non-reduced samples on the same gel,

follow these guidelines:

 Do not run reduced and non-reduced samples in adjacent lanes. The

reducing agent may have a carry-over effect on the non-reduced samples if

they are in close proximity.

 If you are running reduced and non-reduced samples on the same gel, omit

the antioxidant (see page 13). The antioxidant will have a deleterious effect

on the non-reduced sam

ples. The bands will be sharper on NuPAGE

Gels

relative to other gel systems, even without the use of the antioxidant.

Continued on next page

Preparing Buffers for Denaturing Electrophoresis, Continued

Heating Samples

Heat the sample for denaturing electrophoresis (reduced or non-reduced) at

70°C for 10 minutes for optimal results.

Preparing

Running Buffer

Use 1X NuPAGE

SDS Running Buffer for electrophoresis of denatured

samples.

Reducing Conditions

1. Prepare 1,000 mL of Running Buffer as follows:

20X NuPAGE

SDS Running Buffer (MES, MOPS, or Tris-Acetate) 50 mL

Deionized Water 950 mL

Total Volume 1,000 ml

2. Mix thoroughly and set aside 800 mL of the 1X NuPAGE

SDS Running

Buffer for use in the Lower (Outer) Buffer Chamber of the XCell SureLock

™

Mini-Cell.

3. Add 500 μL of NuPAGE

Antioxidant to 200 mL of 1X NuPAGE

SDS

Running Buffer from Step 1 for use in the Upper (Inner) Buffer Chamber of

the XCell SureLock

™

Mini-Cell just prior to starting electrophoresis. Mix

thoroughly.

Non-Reducing Conditions

1. Prepare 1,000 mL of Running Buffer as follows:

20X NuPAGE

SDS Running Buffer (MES or MOPS) 50 mL

Deionized Water 950 mL

Total Volume 1,000 mL

2. Mix thoroughly. Fill the Upper and Lower Buffer Chamber of the XCell

SureLock

™

Mini-Cell with this Running Buffer.

 If the antioxidant is not added to the Upper Buffer Chamber, reoxidation of

proteins during electrophoresis may cause certain bands to appear more

diffuse.

 Prepare Running Buffer for the upper chamber with the antioxidant no

longer than half an hour before use. Antioxidant diluted in Running Buffer

looses effectiveness over time, resulting in gels that exhibit signs of

reoxidation (slightly fuzzier bands).

 If 0.5 mL of antioxidant is added to the total amount of Running Buffer (for

Upper and Lower Buffer Chambers) by accident, the amount of antioxidant

falls below the effective concentration. Additional antioxidant can be added

to increase the concentration (2.5 mL antioxidant in 1 L Running Buffer),

but this is not recommended because high current is generated and the

antioxidant in the Lower Buffer Chamber is wasted.

Preparing Buffers for Non-Denaturing Electrophoresis

Materials Supplied

by the User

The following reagents are needed to prepare samples for non-denaturing

electrophoresis with NuPAGE

Novex

Tris-Acetate Gels. Ordering

information for pre-mixed buffers is on page 48. If you are preparing your own

buffers, recipes a

re provided on page 51–52.

 Protein sample

 Deionized water

For Sample Preparation

 Novex

Tris-Glycine Native Sample Buffer

For Running Buffer Preparation

 Novex

Tris-Glycine Native Running Buffer

Preparing

Samples for

Instructions are provided below to prepare samples for non-denaturing (native)

gel electrophoresis using the NuPAGE

Novex

Tris-Acetate Gels.

Reagent Volume

Sample x μL

Novex

Tris-Glycine Native Sample Buffer (2X) 5 μL

Deionized Water to 5 μL

Total Volume 10 μL

Do not heat samples for non-denaturing (native) electrophoresis.

Preparing

Running Buffer

Use 1X Novex

Tris-Glycine Native Running Buffer for electrophoresis of

samples under non-denaturing (native) conditions.

1. Prepare 1,000 mL Native Running Buffer as follows:

Novex

Tris-Glycine Native Running Buffer (10X) 100 mL

Deionized Water 900 mL

Total Volume 1,000 mL

2. Mix thoroughly and use 800 mL of this Running Buffer in the Lower and

Upper Buffer Chambers of the XCell SureLock

™

Mini-Cell.

Continued on next page

Electrophoresis of NuPAGE

Gels

Introduction

Instructions are provided below for electrophoresis of NuPAGE

Gels using the

XCell SureLock

™

Mini-Cell. For more information on the XCell SureLock

™

Mini-

Cell, refer to the manual (IM-9003). This manual is available on our website at

www.invitrogen.com or contact Technical Support (see page 56).

If you are using any other electrophoresis m

ini-cell, follow the manufacturer’s

recommendations.

Important

To ensure success with the NuPAGE

Electrophoresis System, remember the

important points listed below:

 Wear protective gloves and safety glasses when handling gels.

 Under no circumstances should Tris-Glycine SDS buffers be used with

NuPAGE

Gels for any denaturing gel electrophoresis (see page 46–47 for

the outcome of your results using incorrect buffers).

 Only use NuPAGE

SDS buffers (see page 12).

 DO NOT BOIL samples. Heat samples at 70C for 10 minutes (see page

15).

 Inner and Outer Buffer Cham

bers must be filled with the recommended

amount of running buffer to prevent excessive heating (see below).

Procedure using

XCell SureLock

™

Mini-Cell

XCell SureLock

™

Mini-Cell require 200 mL for the Upper Buffer Chamber and

600 mL for the Lower Buffer Chamber.

1. Remove the NuPAGE

Gel from the pouch.

2. Rinse the gel cassette with deionized water. Peel off the tape from the

bottom of the cassette.

3. Gently pull the comb out of the cassette in one smooth motion.

4. Rinse the sample wells with 1X NuPAGE

SDS Running Buffer. Invert the

gel and shake to remove the buffer. Repeat two more times.

5. Orient the two gels in the Mini-Cell such that the notched “well” side of the

cassette faces inwards toward the Buffer Core. Seat the gels on the bottom

of the Mini-Cell and lock into place with the Gel Tension Wedge. Refer to

the XCell SureLock

™

Mini-Cell manual (IM-9003) for detailed instructions.

Note: If you are running just one gel, use the plastic Buffer Dam in place of

the second gel cassette to form the Upper Buffer Chamber.

6. Fill the Upper Buffer Chamber with a small amount of the Running Buffer

to check for tightness of seal. If you detect a leak from Upper to the Lower

Buffer Chamber, discard the buffer, reseal the chamber, and check the seal

again.

7. Once the seal is tight, fill the Upper Buffer Chamber (Inner) with the

appropriate 1X Running Buffer. The buffer level must exceed the level of

the wells.

Continued on next page

Electrophoresis of NuPAGE

Gels, Continued

Procedure using

XCell SureLock

™

Mini-Cell,

continued

Note: If you are running reduced samples, remember to fill the Upper Buffer

Chamber with 200 mL of running buffer containing the NuPAGE

Antioxidant (see page 15).

8. Load an ap

propriate volume of sample at the desired protein

concentration onto the gel (see page 10 for recommended loading

volumes).

9. Load appropriate protein molecular weight markers (see page 48 for

ordering information).

10. Fill t

he Lower (Outer) Buffer Chamber with 600 mL of the appropriate 1X

Running Buffer.

Electrophoresis

Conditions

Run your gels according to the following table:

Gel Type Voltage Expected Current* Run Time

NuPAGE

Novex

Bis-Tris Gels

with MES SDS Running Buffer

200 V constant† Start: 110–125 mA/gel

End: 70–80 mA/gel

35 minutes

NuPAGE

Novex

Bis-Tris Gels

with MOPS SDS Running Buffer

200 V constant† Start: 100–115 mA/gel

End: 60–70 mA/gel

50 minutes

NuPAGE

Novex

Tris-Acetate

Gels

150 V constant Start: 40–55 mA/gel

End: 25–40 mA/gel

1 hour

NuPAGE

Novex

Tris-Acetate

Native Gels

150 V constant Start: 18 mA/gel

End: 7 mA/gel

~2 hours

Run times

may vary

† Recommended voltage for 9 and 17-well gels is 150–175 volts

Procedure for

NuPAGE

Turbo

Protocol

An optional accelerated process is available for achieving excellent separation

and resolution in only 25 minutes when using NuPAGE

Novex

Bis-Tris Gels

with MES SDS Running Buffer.

The voltage is set at 250 V constant with an expected current of 130–150 mA per

gel, and a run time of 25 minutes.

Continued on next page

Opening Novex

Pre-Cast Gel Cassettes

Removing the Gel

after

electrophoresis

1. After electrophoresis is complete, shut off the power, disconnect electrodes,

and remove gel(s) from the XCell SureLock

™

Mini-Cell.

2. Separate each of the three bonded sides of the cassette by inserting the Gel

Knife into the gap between the two plastic plates that make up the cassette.

The notched (“well”) side of the cassette should face up.

3. Push down gently on the knife handle to separate the plates. Repeat on

each side of the cassette until the plates are completely separated.

Caution: Use caution while inserting the Gel Knife between the two plates

to avoid excessive pressure on the gel.

4. Carefully remove and discard the top plate, allowing the gel to rest on the

bottom (slotted) plate.

5. If blotting, proceed to page 34 without removing the gel from the bottom

plate.

6. If staining, remove the gel from the plate by one of the methods:

 Use the sharp edge of the Gel Knife to remove the gel foot from the

bottom of the gel. Hold the Gel Knife at a 90° angle, perpendicular to

the gel a

nd the slotted half of the cassette. Push down on the knife, and

then repeat the motion across the gel to cut off the entire foot. Hold the

plate and gel over a container with the gel facing downward and use

the knife to carefully loosen one lower corner of the gel and allow the

gel to peel away from the plate.

 Hold the plate and gel over a container with the gel facing downward.

Gently push the Gel Knife through the slot in the cassette, until the gel

peels away from the plate. Cut the gel foot off of the gel after fixing and

staining, but before drying.

7. Fix and stain the gel as described on pages 20–27.

Silver Staining

Introduction

Instructions are provided below for silver staining the NuPAGE

Gels using the

SilverQuest

™

Silver Staining Kit and the SilverXpress

Silver Staining Kit (see

page 49 for ordering information).

If you are using any other silver staining kit, follow the manufacturer’s

recommendations.

The NuPAGE

system is more effective in reducing proteins and maintaining

proteins in their reduced state. This may cause any minor contaminants present

in the protein to be more visible under the sensitive silver staining techniques

with the NuPAGE

system than in other systems.

Materials Supplied

by the User

You will need following items for silver staining your gel (see pages 48–49 for

ordering information on Invitrogen products):

 Staining container

 Rotary Shaker

 Ult

rapure water (>18 megohm/cm resistance recommended)

 Teflon coated stir bars

 Disposable 10 mL pipettes

 Clean glass bottles for reagent preparation

 Graduated glass cylinders

 Protein molecular weight markers (Mark 12

™

Unstained Standard,

recommended)

For SilverQuest

™

Silver Staining

 Ethanol

 Fixative (40% ethanol, 10% acetic acid)

For SilverXpress

Silver Staining

 Methanol

 Acetic acid

For optimal silver staining results, follow these guidelines:

 Be sure to wear clean gloves that have been rinsed with deionized water

while handling gels

 Use clean containers and designate these containers for silver staining

purposes only

 Make sure the size of the container permits free movement of the gel

during shaking and complete immersion in solution while staining

 Do not touch the gel with bare hands or metal objects and do not put

pressure on gels while handling or changing solutions

 Use teflon coated stir bars and clean glass containers to prepare reagents

 Avoid cross contamination of kit reagents

 Use freshly made solutions

Continued on next page

Silver Staining, Continued

Preparing

Solutions for

SilverQuest

™

Silver Staining

Use the reagents provided in the SilverQuest

™

Silver Staining Kit to prepare the

following solutions for staining:

 Sensitizing solution

Ethanol 30 mL

Sensitizer 10 mL

Ultrapure water to 100 mL

 Staining solution

Stainer 1 mL

Ultrapure water to 100 mL

 Developing solution

Developer 10 mL

Developer enhancer 1 drop

Ultrapure water to 100 mL

Note: You may prepare all solutions immediately before starting the staining

protocol or prepare them as you proceed to the next step.

SilverQuest

™

Microwave Silver

Staining Protocol

The microwave protocol for silver staining NuPAGE

Gels with SilverQuest

™

Silver Staining Kit is provided below. For the Basic Protocol and more details on

the staining procedure, refer to the SilverQuest

™

Silver Staining Kit Manual

(IM-6070). This manual is available on our website at www.invitrogen.com or

contact Technical Support (see page 56).

Use 100 m

L of each solution for each 1.0 mm thick, 8 cm × 8 cm NuPAGE

Gel.

Note: You may have to optimize the staining protocol, if the dimensions of your

gel are not the same as mentioned above.

1. After electrophoresis, place the gel in a clean microwaveable staining tray of

the appropriate size. Rinse the gel briefly with ultrapure water.

2. Place the gel in 100 mL of fixative and microwave at high power (700 watts)

for 30 seconds. Remove the gel from the microwave and gently agitate it for

5 minutes at room temperature. Decant the fixative.

3. Wash the gel with 100 mL of 30% ethanol in a microwave at high power for

30 seconds. Remove the gel from the microwave and gently agitate it for

5 minutes at room temperature on a rotary shaker. Decant the ethanol.

4. Add 100 mL of Sensitizing solution to the washed gel. Microwave at high

power for 30 seconds. Remove the gel from the microwave and place it on a

rotary shaker for 2 minutes at room temperature. Decant the Sensitizing

solution.

Continued on next page

Silver Staining, Continued

SilverQuest

™

Microwave Silver

Staining Protocol,

continued

5. Add 100 mL ultrapure water to the gel. Microwave at high power for

30 seconds. Remove the gel from the microwave and gently agitate it for

2 minutes at room temperature. Decant the water, and repeat the step one

more time.

6. Place the gel in 100 mL of Staining solution. Microwave at high power for

30 seconds. Remove the gel from the microwave and gently agitate it for

5 minutes at room temperature.

7. Decant the Staining solution and wash the gel with 100 mL of ultrapure

water for 20–60 seconds. Do not wash the gel for more than a minute.

8. Place the gel in 100 mL of Developing solution and incubate for 5 minutes

at room temperature with gentle agitation on a rotary shaker. Do not

microwave.

9. Once the desired band intensity is achieved, immediately add 10 mL of

Stopper directly to the gel still immersed in Developing solution and gently

agitate the gel for 10 minutes. The color changes from pink to clear

indicating the end of development.

10. Wash the gel with 100 mL of ultrapure water for 10 minutes. For gel

drying, see page 28.

If you need to destain the gel for mass spectrometry analysis, see the

SilverQuest

™

Silver Staining Kit Manual (IM-6070).

Preparing

Solutions for

SilverXpress

Silver Staining

Prepare the reagents as described below. If you are staining two gels, double

the reagent volumes.

 Fixing solution

Methanol 100 ml

Acetic Acid 20 ml

Ultrapure water to 200 mL

 Sensitizing solution

Methanol 100 ml

Sensitizer 5 ml

Ultrapure water to 200 mL

 Staining solution

Stainer A 5 ml

Stainer B 5 ml

Ultrapure water 90 ml

 Developing Solution

Developer 5 ml

Ultrapure water 95 ml

Continued on next page

Silver Staining, Continued

SilverXpress

Silver Staining

Protocol

The following staining procedure is for 1 mm NuPAGE

Gels. If you are using

1.5 mm thick NuPAGE

Gels, double the incubation time.

For gel drying, see page 28.

Note: Gels m

ay be stored in the second Sensitizing Solution overnight, if

desired.

NuPAGE

Gel Type Step Solution Vol/Gel

Tris-Acetate Gel Bis-Tris Gel

1 Fix the gel in Fixing Solution. 200 ml 10 minutes 10 minutes

2A 100 ml 10 minutes 30 minutes

Decant the Fixing Solution

and incubate the gel in two

changes of Sensitizing

Solution.

100 ml 10 minutes 30 minutes

3A 200 ml 5 minutes 10 minutes

Decant the Sensitizing

Solution and rinse the gel

twice with ultrapure water.

200 ml 5 minutes 10 minutes

4 Incubate the gel in Staining

Solution.

100 ml 15 minutes 15 minutes

5A 200 ml 5 minutes 5 minutes

Decant the Staining Solution

and rinse the gel twice with

ultrapure water.

200 ml 5 minutes 5 minutes

6 Incubate the gel in

Developing Solution.

100 ml 3–15 minutes 3–15

minutes

7 Add the Stopping Solution

directly to the gel when the

desired staining intensity is

reached.

5 ml 10 minutes 10 minutes

8A 200 ml 10 minutes 10 minutes

8B 200 ml 10 minutes 10 minutes

Decant the Stopping Solution

and wash the gel three times

in ultrapure water.

200 ml 10 minutes 10 minutes

Molecular Weight

Calibration

Guidelines and apparent molecular weight values for the Novex

protein

molecular weight standards in the NuPAGE

buffer system is provided on

pages 43–44.

Coomassie Staining

Introduction

Instructions are provided below for Coomassie staining of NuPAGE

Gels

using the SimplyBlue

™

SafeStain, Colloidal Blue Staining Kit, and Coomassie R-

250.

If you are using any other Coomassie staining kit, follow the manufacturer’s

recommendations.

If you are staining low molecular weight peptides (<2.5 kDa), we recommend

fixing the gel in 5% glutaraldehyde and 50% methanol for one hour and then

follow the instructions in the Colloidal Blue Staining Kit Manual (IM-6025) for

small peptides.

Materials Supplied

by the User

You will need the following items for staining your gel (see page 49 for

ordering information on Invitrogen products):

 SimplyBlue

™

SafeStain

 Colloidal Blue Staining Kit

 Staining container

 Ultrapure water or deionized water

 Orbital Shaker

 Protein molecular weight standards (see page 45 for ordering information)

 Microwave oven and 20% NaCl (if using SimplyBlue

™

SafeStain microwave

protocol, see page 25)

 Met

hanol and acetic acid (if using Colloidal Blue Staining Kit, see page 26)

For Coom

assie R-250 staining

 0.1% Coomassie R-250 in 40% ethanol and 10% acetic acid

 Destaining Solution (10% ethanol and 7.5% acetic acid)

 Microwave oven (if using Coomassie R-250 microwave protocol, see

page 27)

ontinued on next page

Coomassie Staining, Continued

SimplyBlue

™

SafeStain

Microwave

Protocol

The microwave protocol for staining NuPAGE

Gels with SimplyBlue

™

SafeStain is provided below. For the Basic Protocol and more details on the

staining procedure, refer to the SimplyBlue

™

SafeStain Manual (IM-6050). This

manual is available on our website at www.invitrogen.com or contact Technical

Support (see page 56).

The procedure is written for 1.0 mm thick mini-gels.

After electrophoresis, follow the instructions below:

Caution: Use caution while using the stain in a m

icrowave oven. Do not

overheat the staining solutions.

1. Place the gel in a loosely covered container containing 100 mL of ultrapure

water and microwave on High (950 to 1100 watts) for 1 minute until the

solution is close to boiling.

2. Gently shake the gel on an orbital shaker or rocker for 1 minute. Shake the

gel for 2 minutes for 1.5 mm thick gels. Discard the water.

3. Repeat Steps 1 and 2 two more times.

4. Add 20 mL SimplyBlue

™

SafeStain the mini-gel (the gel should be

completely covered) and microwave on High for 45 seconds to 1 minute

until the solution almost boils. For 1.5 mm thick gels, use 30 mL of stain

and microwave for 1.5 minutes.

5. Shake the gel on an orbital shaker or rocker for 5 minutes. Shake the gel for

10 minutes for 1.5 mm thick gels. The detection limit after staining is 20 ng

BSA.

6. Wash the gel in 100 mL of ultrapure water for 10 minutes on a shaker. The

detection limit after washing is 10 ng BSA.

7. Add 20 mL of 20% NaCl for at least 5 minutes. The detection limit after the

salt wash is 5 ng BSA. Gels can be kept for several weeks in the salt

solution.

8. For gel-drying, see page 28.

ontinued on next page

Coomassie Staining, Continued

Preparing

Solutions for

Colloidal Blue

Staining

Prepare the reagents as described below. If you are staining two gels, double

the reagent volumes. Note: Be sure to shake Stainer B prior to making the

solution.

 Fixing Solution (Bis-Tris Gels)

Methanol 100 mL

Acetic Acid 20 mL

Ultrapure water to 200 mL

 Staining Solution Tris-Acetate Gel Bis-Tris Gel

Deionized water 55 mL 55 mL

Methanol 20 mL 20 mL

Stainer B 5 mL —

Stainer A 20 mL 20 mL

Colloidal Blue

Staining Kit

Protocol

A brief staining protocol for staining NuPAGE

Gels with the Colloidal Blue

Staining Kit is provided below. For more details on the staining procedure,

refer to the Manual (IM-6025). This manual is available on our website at

www.invitrogen.com or contact Technical Support (see page 56).

Colloidal Blue Staining Kit Protocol for NuPAGE

Novex

Tris-Acetate Gels

1. Incubate the gel in Staining Solution for a minimum of 3 hours and a

maximum of 12 hours at room temperature with gentle shaking.

2. Decant the Staining Solution and add at least 200 mL of deionized water

per gel to the staining container. Gently shake the gel in water for at least

7 hours. The gel background should be clear after 7 hours in water.

3. For gel-drying, see page 28.

Colloidal Blue Staining Kit Protocol for NuPAGE

Novex

Bis-Tris Gels

Note: If you are staining low molecular weight peptides (< 2.5 kDa), we

recommend fixing the gel in 5% glutaraldehyde and 50% methanol for one

hour and then follow the instructions in the Colloidal Blue Staining Kit Manual

(IM-6025) for small peptides.

1. Incubate the gel in Fixing Solution for 10 minutes at room temperature

with gentle shaking.

2. Incubate the gel in this Staining Solution (without Stainer B) for

10 minutes at room temperature with gentle shaking.

3. Add 5 mL Stainer B per gel to the Staining Solution from previous step.

Continue staining for a minimum of 3 hours and a maximum of 16 hours.

4. Decant the Staining Solution and add 200 mL of deionized water per gel

to the staining container. Gently shake the gel in water for at least

7 hours. The gel background should be clear after 7 hours in water.

5. For gelfdrying, see page 28.

Note: NuPAGE

Gels can be left in deionized water for up to 3 days without

significant change in band intensity and background clarity. For long-term

storage (over 3 days), keep the gel in a 20% ammonium sulfate solution at 4°C.

Coomassie Staining, Continued

Coomassie R-250

Microwave

Staining Protocol

The Coomassie staining protocol described below is recommended for staining

NuPAGE

Gels. You may use any Coomassie staining protocol of choice.

1. Prepare the staining solution containing 0.1% Coomassie R-250 in

40% ethanol, 10% acetic acid.

2. After electrophoresis, incubate 1 or 2 gels in a staining container containing

100 mL of staining solution prepared in Step 1.

3. Loosely cover the staining container and heat in a microwave oven at full

power for 1 minute. To prevent hazardous, flammable vapors from

forming, do not allow the solution to boil.

4. Remove the staining container from the microwave oven and gently shake

the gel for 15 minutes at room temperature on an orbital shaker.

5. Decant the stain and rinse the gel once with deionized water.

6. Prepare a destain solution containing 10% ethanol and 7.5% acetic acid.

7. Place one or two stained gels in a staining container containing 100 mL of

destain solution prepared in Step 6.

8. Loosely cover the staining container and heat in a microwave oven at full

power for 1 minute.

9. Gently shake the gel at room temperature on an orbital shaker until the

desired background is achieved.

Note: The NuPAGE

Gels destain faster than other Novex

Gels. To

prevent over destaining of NuPAGE

Gels if destaining overnight, dilute

the destain solution by adding 100 mL of deionized water to 100 mL of the

destain solution in the staining container.

10. For-gel-drying, see page 28.

Molecular Weight

Calibration

Guidelines and apparent molecular weight values for the Novex

protein

molecular weight standards in the NuPAGE

buffer system is provided on

pages 43–44.

SYPRO

Ruby Staining

Introduction

Instructions are provided below for a basic and rapid protocol for Novex

Pre-

Cast Gels (NuPAGE

Novex

Bis-Tris and Tris-acetate gels) for the detection of

proteins, including glycoproteins and phosphoproteins.

Advantages of

SYPRO

Ruby

Staining

SYPRO

Ruby provides the following advantages:

 Linear quantitation range of over three orders of magnitude

 Compatible with subsequent analysis of proteins by Edmanbased

sequencing or mass spectrometry in 1D or 2D format

 Compatible with nondenaturing gels and IEF gels (basic protocol)

Molecular Weight

Calibration

Guidelines and apparent molecular weight values for Novex

protein

molecular weight standards are provided on page 43.

Materials Supplied

by the User

 Staining containers, 1 per gel (see below for details)

 Reagent-grade methanol

 Reagent-grade glacial acetic acid

 Trichloroacetic acid (for IEF gels only)

 Ultrapure water (18 megohm-cm recommended)

 Rotary shaker

 Powder-free latex or vinyl gloves

 Microwave oven (700–1200 W) (optional)

 Water bath set at 80°C (optional)

General considerations for the protocol include the following:

 Perform all fixation, staining, and washing steps with continuous, gentle

agitation (e.g., on an orbital shaker at 50 rpm)

 We recommend polypropylene or polycarbonate containers for staining.

Glass dishes are not recommended. Staining containers should be

meticulously clean to minimize contamination and other artifacts

 For convenience, gels may be left in fix solution overnight or longer

 For convenience, gels may be left in SYPRO

Ruby stain indefinitely

without overstaining, although speckling artifacts tend to increase over

time

 As with any fluorescent stain, cover the gel container during staining and

subsequent wash steps to exclude light

Continued on next page

SYPRO

Ruby Staining, Continued

Preparing

Solutions for

SYPRO

Ruby

Staining

Prepare the reagents as described below. If you are staining two gels, double

the reagent volumes. Increase volumes 1.5-fold for 1.5mm thick gels.

 Fix Solution

Methanol 100 mL

Glacial Acetic Acid 14 mL

Ultrapure water to 200 mL

 Fix Solution for IEF Gels

Methanol 40 mL

Trichloroacetic Acid 10 g

Ultrapure water to 100 mL

 Wash Solution

Methanol 10 mL

Glacial Acetic Acid 7 mL

Ultrapure water to 100 mL

SYPRO

Ruby

Basic Protocol

The basic protocol results in the maximum signal strength and widest linear

dynamic range for staining of denaturing gels, nondenaturing gels, and IEF

gels. Sensitivity is in the 1 ng range for most proteins.

1. After electrophoresis, place the gel into a clean container with 100 mL of Fix

Solution and agitate on an orbital shaker for 30 minutes. Pour off the used

fix solution and repeat once more with fresh Fix Solution.

Note: For IEF Gels, place the gel into a clean container with 100 mL of IEF

Fix Solution and agitate on an orbital shaker for 3 hours. After fixing,

perform 3 washes in ultrapure water for 10 minutes each, before

proceeding to the staining step.

2. Pour off the used fix solution.

3. Add 60 mL of SYPRO

Ruby gel stain to the tray containing the gel. Agitate

on an orbital shaker overnight.

4. Transfer the gel to a clean container and wash in 100 mL of Wash Solution

for 30 minutes. The transfer step helps minimize background staining

irregularities and stain speckles on the gel.

5. Rinse the gel in ultrapure water for 5 minutes. Repeat the rinse a minimum

of one more time to prevent possible corrosive damage to your imager.

Note: If you are staining two gels, double the reagent volumes. Increase

volumes 1.5-fold for 1.5mm thick gels.

Visualization of

SYPRO

Ruby

Stained Gels

Proteins stained with SYPRO® Ruby protein gel stain are readily visualized

using a UV or blue-light source. The use of a photographic camera or CCD

camera and the appropriate filters is essential to obtain the greatest sensitivity.

Continued on next page

SYPRO

Ruby Staining, Continued

SYPRO

Ruby

Rapid Protocol

The rapid protocol is optimized for Invitrogen NuPAGE

gels, and can be

completed in 90 minutes. While the maximum fluorescence signal strength is

lower than for the basic protocol, the results show excellent linearity and low

background, with a lower limit of detection of 0.25 to 1 ng for most proteins.

1. After electrophoresis, place the gel into a clean a microwavable container

with 100 mL of Fix Solution and agitate on an orbital shaker for 15 minutes.

Pour off the used fix solution and repeat once more with fresh Fix Solution.

2. Pour off the used fix solution.

3. Add 60 mL of SYPRO

Ruby gel stain to the tray containing the gel.

4. Microwave 30 seconds, agitate 30 seconds to distribute heat evenly, then

microwave another 30 seconds to reach 80–85°C, and agitate on an orbital

shaker for 5 minutes.

5. Reheat the gel by microwaving a third time for 30 seconds and then agitate

on an orbital shaker for 23 minutes for a total stain time of 30 minutes.

Note: An acceptable alternative to the microwave procedure is to incubate

the gel at 80°C in a shaking water bath for a total of 30 minutes.

6. Transfer the gel to a clean container and wash in 100 mL of Wash Solution

for 30 minutes. The transfer step is necessary to avoid heating the destain

solution, which may reduce stain sensitivity, and also helps minimize

background staining irregularities and stain speckles on the gel.

7. Rinse the gel in ultrapure water for 5 minutes. Repeat the rinse a minimum

of one more time to prevent possible corrosive damage to your imager.

Note: If you are staining two gels, double the reagent volumes. Increase

volumes 1.5-fold for 1.5mm thick gels.

Heat the gel in the microwave on full power in increments of 30–45 seconds,

until the stain reaches 80–85°C.

Do not heat the fixative solution or other methanolic solutions in the

microwave.

Although SYPRO

Ruby stain solution is not flammable, use caution when

microwaving SYPRO

Ruby stain as the solution becomes very hot.

Using SYPRO

Ruby Stain As a

Post-Stain

SYPRO

Ruby stain can be used to post-stain gels stained with other gel stains

such as Pro-Q

Diamond phosphoprotein gel stain, Pro-Q

Emerald 300

glycoprotein gel stain, Pro-Q

Sapphire or InVision

™

oligohistidine-tag gel

stains, Pro-Q

Amber transmembrane protein gel stain, or silver staining (such

as Invitrogen SilverQuest

™

or SilverXpress

stain).

Always use SYPRO

Ruby stain last, as the SYPRO

Ruby signal can dominate

the signal from other stains. SYPRO

Ruby stain does not work well as a post-

stain for colorimetric stains such as Coomassie and silver stains.

Gel Drying

Introduction

Dry gels by passive evaporation (air-drying) or vacuum drying. Vacuum

drying is faster than passive air-drying methods but often results in cracked

gels due to the speed of dehydration.

We recommend drying Novex

Pre-Cast gels using passive air-drying methods

such as DryEase

Mini-Gel Drying System (see below). For applications that

require vacuum drying, follow the recommendations on page 33 to minimize

cracking of the gels.

Do not leave Coomassie stained gels in Gel-Dry

™

solution (or any equilibration

solution containing >20% alcohol) for more than 5 minutes. Gels left in this

solution for longer than 5 minutes lose band intensity and result in decreased

sensitivity.

Materials Supplied

by the User

You will need the following items for drying your gel (see page 48 for ordering

information on Invitrogen products):

 DryEase

Mini-Gel Drying System

 Gel-Dry

™

Drying Solution (or prepare your own gel drying solution

containing 30% methanol and 5% glycerol)

 StainEase

Gel Staining Tray

DryEase

Mini-Gel

Drying System

A brief gel drying protocol using the DryEase

Mini-Gel Drying System is

provided below. For more details on this system, refer to the DryEase

Mini-

Gel Drying System manual (IM-2380). This manual is available for download

from our website at www.invitrogen.com or contact Technical Support (see

page 56).

Wear gloves while handling gels and gel drying solution.

1. After all staining and destaining steps are complete, wash the destained

gel(s) three times for two minutes ea

ch time in deionized water (50 mL per

mini-gel) on a rotary shaker.

2. Decant the water and add fresh Gel-Dry

™

Drying Solution (35 mL per mini-

gel).

3. Equilibrate the gel in the Gel-Dry

™

Drying Solution by shaking the gel for

15–20 minutes in the StainEase

Gel Staining Tray or in a round container.

Note: Do not equilibrate Coomassie stained gels in the Gel-Dry

™

Drying

Solution for more than 5 minutes to avoid losing band intensity.

4. Cut any rough edges off the gel (including the wells and the gel foot) using

the Gel Knife or a razor blade.

5. Remove 2 sheets of cellophane per gel from the package.

6. Immerse one sheet of cellophane in the Gel-Dry

™

Drying Solution. Allow

15–20 seconds for complete wetting before adding additional sheets. Do not

soak the cellophane sheets for more than 2 minutes.

Continued on next page

Gel Drying, Continued

DryEase

Mini-Gel

Drying System,

continued

7. Place one side of the DryEase

Gel Drying Frame with the corner pin facing

up, on the DryEase

Gel Drying Base.

8. Center a piece of pre-wetted cellophane from Step 5 over the base/frame

combination, so the cellophane lays over the inner edge of the frame.

9. Lay the gel on the center of the cellophane sheet making sure no bubbles are

trapped between the gel and the cellophane. Add some Gel-Dry

™

Drying

Solution to the surface of the cellophane, if necessary.

10. Carefully lay the second sheet of cellophane over the gel so that no bubbles are

trapped between the cellophane and the gel. Add some Gel-Dry

™

Drying

Solution if necessary. Gently smooth out any wrinkles in the assembly with a

gloved hand.

11. Align the remaining frame so that its corner pins fit into the appropriate holes

on the bottom frame. Push the plastic clamps onto the four edges of the

frames.

12. Lift the frame assembly from the DryEase

Gel Drying Base and pour off the

excess solution from the base.

13. Place the gel dryer assembly upright on a benchtop. Be careful to avoid drafts

as they can cause an uneven rate of dying which leads to cracking. Drying

takes between 12–36 hours depending on humidity and gel thickness.

14. When the cellophane is dry to touch, remove the gel/cellophane sandwich

from the drying frame. Trim off the excess cellophane.

15. Press the dried gel(s) between the pages of a notebook under light pressure for

approximately 2 days so they remain flat for scanning, photography, display,

and overhead projection.

Continued on next page

Gel Drying, Continued

Vacuum Drying

General guidelines are provided below to minimize cracking during vacuum

drying of gels. For detailed instructions, follow the manufacturer’s

recommendations.

Handle Gels with Care:

Remove the gel from the cassette without breaking or tearing the edges. Small

nicks or tears can act as a starting point for cracking. Remove the gel wells and

foot off the bottom of the gel with a Gel Knife or a razor blade as described on

page 19. Use the StainEase Staining Tray for staining and destaining gels. This

tray is designed to facilitate the solution changing process without handling of

gels.

Use a Gel Drying Solution:

We recommend equilibrating the gel in a gel drying solution such as Gel-Dry

™

Gel Drying Solution for 10–30 minutes at room temperature with gentle

shaking on an orbital shaker before drying the gel. Gel-Dry

™

Gel Drying

Solution contains a proprietary non-glycerol component to effectively regulate

the rate of drying and prevent cracking. The gel drying solution does not

interfere with autoradiography.

To prepare your own gel drying solution, prepare a solution containing

30% methanol and 5% glycerol.

Note: Do not incubate Coomassie stained gels in gel drying solution for more

than 5 minutes as the bands may fade.

Remove Air Bubbles:

Remove any air bubbles that may be trapped between the paper, gel, and

plastic wrap by rolling a small glass pipette over the gel. Use additional gel

drying solution to remove any air bubbles.

Use Proper Gel Dryer Set-up:

Place gel on the gel dryer with the plastic wrap facing up. Make sure the

vacuum pump is in working condition, and properly set up to form a tight seal

when on. Use drying conditions for polyacrylamide gels, with the temperature

increasing to a set value and holding for the duration of the drying cycle. We

recommend drying mini-gels at 80C for 2 hours.

Ensure Gel is Completely Dry:

The gel will crack if the vacuum seal of the heated gel dryer is broken prior to

complete drying of the gel. To ensure the gel is completely dried before

releasing the vacuum seal, follow these tips :

 Check the temperature of the gel

The temperature of the dried gel should be the same as the temperature of

the surrounding gel drying surface. If the temperature of the dried gel is

cooler, then the gel is not completely dried.

 Check for moisture in the tubing connecting the gel dryer to the vacuum

pump

The gel is not completely dried if there is residual moisture in the tubing

and additional drying time is required.

Western Blotting

Introduction

After performing electrophoresis, proteins can be transferred to membranes for

subsequent analysis. Methods of transfer include wet, semi-wet, semi-dry, and

dry blotting.

Semi-dry blotting can be performed with the Novex

Semi-Dry Blotter or other

semi-dry blotter. For details on performing semi-dry blotting, see page 39 or

refer to the manual for the Novex

Semi-Dry Blotter (25-0911).

Dry blotting is performed with the iBlot

Gel Transfer Device. Refer to the

manual for the iBlot

Dry Blotting System (25-0949) for details.

Instructions are provided below for semi-wet blotting of NuPAGE

Gels using

the XCell II

™

Blot Module. For more information on the XCell II

™

Blot Module,

refer to the manual (IM-9051) available at www.invitrogen.com or contact

Technical Support (see page 56).

NuPAGE

Antioxidant

The NuPAGE

Antioxidant is added to the transfer buffer when blotting reduced

proteins to prevent reoxidation and maintain proteins in a reduced state (see

page 13).

The anode electrochemistry is the major cause of reoxidation during blotting,

though proteins are oxidized at a slower rate in the neutral pH environment of

the NuPAGE

blotting system compared to higher pH blotting systems.

NuPAGE

Transfer

Buffer

We recommend using the NuPAGE

Transfer Buffer for western transfer of

NuPAGE

Gels to maintain the neutral pH environment established during

NuPAGE

electrophoresis.

The NuPAGE

Transfer Buffer protects against modification of amino acid side

chains and is compatible with N-terminal protein sequencing using Edman

degradation.

Materials Supplied

by the User

In addition to the XCell II

™

Blot Module, the following reagents are needed for

blotting your gel (see page 48–49 for ordering information on Invitrogen

products):

 Bl

otting membranes

 Filter paper

 Methanol (if using PVDF membranes)

 XCell II

™

Blot module

 NuPAGE

Transfer Buffer

 NuPAGE

Antioxidant for reduced samples

 MagicMark

™

Western Protein Standard

 Deionized water

Continued on next page

Western Blotting, Continued

Preparing

NuPAGE

Transfer

Buffer

For blotting NuPAGE

Gels, use 1X NuPAGE

Transfer Buffer. If you are

preparing your own transfer buffer see page 53 for a recipe.

Prep

are 1,000 mL of Transfer Buffer (20X) as follows:

Reduced Samples

Non-Reduced Samples

NuPAGE

Transfer Buffer (20X) 50 mL 50 mL

NuPAGE

Antioxidant 1 mL —

Methanol 100 mL * 100 mL *

Deionized Water 849 mL 850 mL

Total Volume 1,000 mL 1,000 mL

*NuPAGE

Transfer Buffer with 10% methanol provides optimal transfer of a

single gel in the blot module. If you are transferring two gels in the blot module,

increase the methanol content to 20% to ensure efficient transfer of both gels.

Preparing Blotting

Pads

Use about 700 mL of 1X NuPAGE

Transfer Buffer to soak the pads until they

are saturated. Remove the air bubbles by squeezing the pads while they are

submerged in buffer. Removing the air bubbles is essential as they can interfere

with the transfer of biomolecules if not removed.

Preparing

Transfer

Membrane and

Filter Paper

Cut the transfer membrane and filter paper to the dimensions of the gel, or use

Novex

pre-cut membrane/filter paper sandwiches (see page 48 for ordering

information.

 PVDF membrane—Pre-wet PVDF membrane for 30 seconds in methanol,

ethanol (95%), or isopropanol. Briefly rinse in deionized water, then place

in a shallow dish with 50 mL of 1X NuPAGE

Transfer Buffer for several

minutes.

 Nitrocellulose—Place the membrane directly into a shallow dish

containing 50 mL of 1X NuPAGE

Transfer Buffer for several minutes.

 Filter paper—Soak the filter paper briefly in 1X NuPAGE

Transfer Buffer

immediately prior to use.

 Gel—Use the gel immediately following the run. Do not soak the gel in

transfer buffer.

Continued on next page

Western Blotting, Continued

Western Transfer

Using the XCell II

™

Blot Module

Wear gloves while performing the blotting procedure to prevent contamination

of gels and membranes, and exposure to irritants commonly used in

electrotransfer.

Transferring One Gel

1. After opening the gel cassette as described on page 19 remove wells with

the Gel Knife.

2. Place a piece of pre-soaked filter paper on top of the gel, with the edge

bove the slot in the bottom of the cassette (leaving the foot of the gel

uncovered). Keep the filter paper saturated with the transfer buffer and

remove all trapped air bubbles by gently rolling over the surface using a

glass pipette as a roller.

3. Turn the plate over so the gel and filter paper are facing downwards over a

gloved hand or clean flat surface.

4. Use the Gel Knife to push the foot out of the slot in the plate, and separate

the gel from the plate.

5. When the gel is on a flat surface, cut the foot off the gel with the Gel Knife.

6. Wet the surface of the gel with transfer buffer and position the pre-soaked

transfer membrane on the gel, ensuring all air bubbles have been removed.

7. Place another pre-soaked filter paper on top of the membrane. Remove any

trapped air bubbles.

8. Place two soaked blotting pads into the cathode (–) core of the blot module.

The cathode core is the deeper of the two cores and the corresponding

electrode plate is a darker shade of gray. Carefully pick up the

gel/membrane assembly and place on blotting pad such that the gel is

closest to the surface of the cathode core (see Figure 1, next page).

9. Add enough pre-soaked blotting pads to raise the assembly 0.5 cm over the

edge of cathode core. Place the anode (+) core on top of the pads. The

gel/membrane assembly should be held securely between the two halves

of the blot module ensuring complete contact of all components.

10. Position the gel/membrane assembly and blotting pads in the cathode core

of the XCell II

™

Blot Module to fit horizontally across the bottom of the

unit. There should be a gap of approximately 1 cm at the top of the

electrodes when the pads and assembly are in place.

11. Hold the blot module together firmly and slide it into the guide rails on the

Lower Buffer Chamber. The blot module fits into the unit one way, with the

(+) sign at the upper left hand corner of the blot module, and the inverted

gold post fitting into the connector on the right side of the Lower Buffer

Chamber.

12. Place the Gel Tension Wedge so that its vertical face is against the blot

module. Lock the Gel Tension Wedge by pulling the lever forward.

Continued on next page

Western Blotting, Continued

Western Transfer

Using the XCell II

™

Blot Module,

continued

13. Fill the blot module with 1X NuPAGE

Transfer Buffer until the

gel/membrane assembly is covered in Transfer Buffer. To avoid generating

extra conductivity and heat, do not fill the chamber all the way to the top.

14. Fill the Lower Buffer Chamber with deionized water by pouring

approximately 650 mL in the gap between the front of the blot module and

the front of the Lower Buffer Chamber. The water level should reach

approximately 2 cm from the top of the Lower Buffer Chamber. This serves

to dissipate heat produced during the run.

15. Place the lid on top of the unit.

16. With the power turned off, plug the red and black leads into the power

supply. Refer to Recommended Transfer Conditions on the next page for

transfer conditions.

Transferring Two Gels in One Blot Module

1. Prepare 1X NuPAGE

Transfer Buffer containing 20% methanol as

described on page 35.

2. Rep

eat Steps 1–7 (previous page) twice to make two gel/membrane

sandwiches.

3. Place two pre-soaked pads on cathode shell of blot module. Place the first

gel/membrane assembly on the pads such that the gel faces the cathode

plate. (See Figure 2).

4. Add another pre-soaked blotting pad on top of first gel/membrane

assembly.

5. Position second gel/membrane assembly on top of blotting pad with the

gel facing the cathode side.

6. Proceed with Steps 8–13 from Transferring One Gel.

7. Refer to Recommended Transfer Conditions on the next page for transfer

conditions.

Figure 1 Figure 2

Filter Paper

Gel

Transfer Membrane

Filter Paper

Blotting Pad

Cathode Core (-)

Blotting Pad

Filter Paper

Transfer Membrane

Second Gel

Blotting Pad

Filter Paper

Blotting Pad

Filter Paper

Transfer Membrane

First Gel

Filter Paper

Blotting Pad

Cathode Core (-)

Continued on next page

Western Blotting, Continued

Recommended

Transfer

Conditions

The transfer conditions for NuPAGE

Gels using the XCell II

™

Blot Module are

listed in the table below.

Note: The expected current listed in the table is for transferring one gel. If you

are transferring two gels in the blot module, the expected current will double.

Gel Transfer Buffer Membrane Power Conditions

NuPAGE

Novex

Bis-Tris

Gel

1X NuPAGE

Transfer

Buffer with 10% methanol*

0.1% NuPAGE

Antioxidant

for reduced samples

Nitrocellulose or

PVDF

30 Volts constant for 1 hour

Expected Current

Start: 170 mA

End: 110 mA

NuPAGE

Novex

Tris-

Acetate Gel

1X NuPAGE

Transfer

Buffer with 10% methanol*

0.1% NuPAGE

Antioxidant

for reduced samples

Nitrocellulose or

PVDF

30 Volts constant for 1 hour

Expected Current

Start: 220 mA

End: 180 mA

*NuPAGE

Transfer Buffer with 10% methanol provides optimal transfer conditions when blotting a single gel in a blot

module. If transferring two gels with the blot module, increase the methanol content to 20% to ensure even and efficient

transfer of both gels.

Alternate Transfer

Buffers

The NuPAGE

Transfer Buffer (with NuPAGE Antioxidant for reduced

samples) is the optimal buffer for western transfer of NuPAGE

gels. However,

NuPAGE

Gels can be blotted with the Tris-Glycine Transfer Buffer (1X) or TBE

Transfer Buffer (1/2X). The NuPAGE

Antioxidant is less effective when added

to the Tris-Glycine and TBE buffers due to the pH.

Carbonate and CAPS transfer buffers are not recommended for blotting of

NuPAGE

Novex

Pre-Cast Gels. The NuPAGE

Antioxidant is ineffective at

pH >9 and will not work when used with the Carbonate or CAPS transfer

buffers.

Continued on next page

Western Blotting, Continued

Semi-Dry Blotting

of NuPAGE

Novex

Bis-Tris

Gels

The NuPAGE

Novex

Bis-Tris Gels do not transfer as efficiently with semi-dry

blotting compared to blotting with the XCell II

™

Blot Module. If you decide to

use semi-dry blotting for NuPAGE

Novex

Bis-Tris Gels, follow the protocol

provided below to ensure efficient transfer of proteins.

1. Prepare 100 mL of 2X NuPAGE

Transfer Buffer as follows:

NuPAGE

Transfer Buffer (20X) 10.0 mL

NuPAGE

Antioxidant (for reduced sample) 0.1 mL

Methanol 10.0 mL

Deionized Water to 100

Total Volume 100 mL

2. Soak the filter paper and transfer membrane in 2X NuPAGE

Transfer

Buffer.

 If you are using Novex

pre-cut membrane/filter sandwiches, use three

pieces of filter paper (0.4 mm/filter in thickness) on each side of the

gel/membrane assembly.

 If you are not using the Novex

pre-cut membrane/filter sandwiches,

use two pieces of thick filter paper) on each side of the gel/membrane

assembly.

3. Prepare 100 mL of 2X NuPAGE

Transfer Buffer ((without methanol)

4. Equilibrate the gel in 2X NuPAGE

Transfer Buffer (without methanol) for

10 minute with gentle agitation.

Note: For transfer of large proteins (>100 kDa), equilibrate the gel with

2X NuPAGE

Transfer Buffer (without methanol) with 0.02–0.04% SDS.

5. Assemble the gel/membrane/filter paper sandwich on top of the anode

plate (+) as follows:

6. Perform the transfer at 20 V for 30–60 minutes if using the Novex

Semi-

Dry Blotter. Perform the transfer at 15 V constant for 15 minutes if you are

using the Bio-Rad Trans-Blot

Semi-Dry Transfer Cell. For other semi-dry

transfer cells, follow the manufacturer’s recommendations.

Using ZOOM

Gels

ZOOM

Gels

ZOOM

Gels are used for 2D analysis of proteins following isoelectric focusing

of IPG strips. ZOOM

Gels are 1.0 mm thick, and contain an IPG well and a

molecular weight marker well. The IPG well is designed to accommodate a

7.0 cm IPG strip.

Two types of ZOOM

Gels are available (see page 48 for ordering information)

 NuPAGE

Novex

4–12% Bis-Tris ZOOM

Gel

 Novex

4–20% Tris-Glycine ZOOM

Gel

Second

Dimension

Electrophoresis

The second dimension electrophoresis procedure involves reducing and

alkylating the proteins focused on your IPG strip in equilibration buffer,

loading the strip on your second dimension gel, and performing SDS-PAGE.

Materials

Supplied by the

User

You will need the following items for running ZOOM

Gels (see page 48–49 for

ordering information on Invitrogen products):

 4X

NuPAGE

LDS Sample Buffer

 NuPAGE

Sample Reducing Agent

 NuPAGE

Novex

4–12% Bis-Tris ZOOM

Gel or Novex

4–20% Tris-

Glycine ZOOM

Gel

 Appropriate running buffer depending on the type of gel you are using

 0.5% agarose solution

 Iodoacetamide

 Plastic flexible ruler or thin weighing spatula

 15 mL conical tubes

 Water bath set at 55C or 65C

 XCell SureLock

™

Mini-Cell

 Protein molecular weight marker

Equilibrating the

IPG Strip

1. Dilute 4X NuPAGE

LDS Sample Buffer to 1X with deionized water.

2. Add 500 μL of the NuPAGE

Sample Reducing Agent to 4.5 mL of the

1X NuPAGE

LDS Sample Buffer from Step 1 in a 15 mL conical tube. Place

one IPG strip in this conical tube for equilibration.

3. Incubate for 15 minutes at room temperature. Decant the Reducing Solution.

4. Prepare 125 mM Alkylating Solution fresh by adding 116 mg of

iodoacetamide to 5 mL of 1X NuPAGE

LDS Sample Buffer from Step 1.

5. Add 5 mL of Alkylating Solution (from Step 4) to the conical tube containing

the IPG strip. Incubate for 15 minutes at room temperature.

6. Decant the Alkylating Solution and proceed to SDS-PAGE, next page. Use

the equilibrated IPG strip immediately for second dimension.

Continued on next page

Using ZOOM

Gels, Continued

SDS-PAGE

A protocol for separating proteins in an IPG strip by SDS-PAGE with ZOOM

Gels and the XCell SureLock

™

Mini-Cell is provided below. You may download

the XCell SureLock

™

Mini-Cell manual from our website at

www.invitrogen.com or contact Technical Support (see page 56).

1. Prep

are 0.5% agarose solution in the appropriate running buffer and keep it

warm (55–65C) until you are ready to use the agarose solution.

2. If the molecular weight marker well is bent, straighten the well using a gel

loading tip.

3. Cut the plastic ends of the IPG strip flush with the gel. Do not cut off any

portions of the gel.

4. Slide the IPG strip into the ZOOM

Gel well.

5. Align the IPG strip properly in the ZOOM

Gel well using a thin plastic

ruler or a weighing spatula. Avoid introducing any air bubbles while

sliding the strip.

6. Pour ~ 400 μL of 0.5% agarose solution into the ZOOM

Gel well

containing the IPG strip. Take care that the agarose solution does not

overflow into the molecular weight marker well.

7. Assemble the gel cassette/Buffer Core sandwich as described in the XCell

SureLock

™

Mini-Cell manual.

Note: If you are running just one gel, use the plastic Buffer Dam in place of

the second gel cassette to form the Upper Buffer Chamber.

Do not use the ZOOM

IPGRunner

™

Core for electrophoresis of the second

dimension gel. You must use the Buffer Core supplied with the XCell

SureLock

™

Mini-Cell.

8. Fill the Upper Buffer Chamber with a small amount of Running Buffer, and

make sure there are no leaks.

9. Fill the Upper Buffer Chamber and Lower Buffer Chamber with the

appropriate Running Buffer.

10. Load molecular weight standards in the marker well.

11. Place the XCell SureLock

™

Mini-Cell lid on the Buffer Core. With the power

on the power supply turned off, connect the electrode cords to the power

supply [red to (+) jack, black to (–) jack].

12. Perform electrophoresis at 200 V for 40 minutes for NuPAGE

Novex

Bis-

Tris ZOOM

Gels or at 125 V for 90 minutes for Novex

Tris-Glycine

ZOOM

Gels.

13. At the end of electrophoresis, turn off the power and disassemble the gel

cassette/Buffer Core sandwich assembly as described in the XCell

SureLock

™

Mini-Cell manual.

14. Proceed to staining the second dimension gel using an appropriate method

for the type of gel and sample amount.

Calibrating Protein Molecular Weight

Introduction

The molecular weight of a protein can be determined based upon its relative

mobility by constructing a standard curve with protein standards of known

molecular weights.

The protein mobility in SDS-PAGE gels is dependent on the

 Length of the protein in its fully denatured state,

 SDS-PAGE buffer systems, and

 Secondary structure of the protein.

The same molecular weight standard may have slightly different mobility,

resulting in different apparent molecular weight when run in different SDS-

PAGE buffer systems.

If you are using the Novex

protein molecular weight standards, see the

apparent molecular weights of these standards in the NuPAGE

Gels listed on

the next page to determine an apparent molecular weight of your protein.

Protein Secondary

Structure

When using SDS-PAGE for molecular weight determination, slight deviations

from the calculated molecular weight of a protein (calculated from the known

amino acid sequence) can occur due to the retention of varying degrees of

secondary structure in the protein, even in the presence of SDS. This

phenomenon is observed in highly organized secondary structures (collagens,

histones, or highly hydrophobic membrane proteins) and in peptides, where

the effect of local secondary structure becomes magnified relative to the total

size of the peptide.

Buffer Systems

Slight differences in protein mobilities also occur when the same proteins are

run in different SDS-PAGE buffer systems. Each SDS-PAGE buffer system has a

different pH, which affects the charge of a protein and its binding capacity for

SDS. The degree of change in protein mobility is usually small in natural

proteins but more pronounced with “atypical” or chemically modified proteins

such as pre-stained standards.

Continued on next page

Calibrating Protein Molecular Weight, Continued

Assigned Apparent

Molecular Weights

The apparent molecular weight values for the Novex

protein standards in several

buffer systems including the NuPAGE

buffer system are provided below. Use the

one that matches your gel for the most accurate calibration of your protein.

The following charts summarize the approximate molecular weight values for the

Novex

protein molecular weight standards when run in the NuPAGE

Buffer

System. You may generate calibration curves in your lab with any other

manufacturer’s standards.

Novex

Sharp Pre-stained

Protein Standard

NuPAGE

(4–12%)

Bis-Tris/MES

NuPAGE

(4–12%)

Bis-Tris/MOPS

NuPAGE

(3–8%)

Tris-Acetate

Band 1 260 kDa 260 kDa 260 kDa

Band 2 160 kDa 160 kDa 160 kDa

Band 3 110 kDa 110 kDa 110 kDa

Band 4 80 kDa 80 kDa 80 kDa

Band 5 60 kDa 60 kDa 60 kDa

Band 6 50 kDa 50 kDa 50 kDa

Band 7 40 kDa 40 kDa 40 kDa

Band 8 30 kDa 30 kDa 30 kDa

Band 9 20 kDa 20 kDa

Band 10 15 kDa 15 kDa

Band 11 10 kDa 10 kDa

Band 12 3.5 kDa

Mark 12

™

Unstained Standard NuPAGE

(4–12%)

Bis-Tris/MES

NuPAGE

(4–12%)

Bis-Tris/MOPS

NuPAGE

(3–8%)

Tris-Acetate

Myosin 200 kDa 200 kDa 200 kDa

 Galactosidase

116.3 kDa 116.3 kDa 116.3 kDa

Phosphorylase B 97.4 kDa 97.4 kDa 97.4 kDa

Bovine Serum Albumin 66.3 kDa 66.3 kDa 66.3 kDa

Glutamic Dehydrogenase 55.4 kDa 55.4 kDa 55.4 kDa

Lactate Dehydrogenase 36.5 kDa 36.5 kDa 36.5 kDa

Carbonic Anhydrase 31 kDa 31 kDa 31 kDa

Trypsin Inhibitor 21.5 kDa 21.5 kDa N/A

Lysozyme 14.4 kDa 14.4 kDa N/A

Aprotinin 6 kDa 6 kDa N/A

Insulin B Chain 3.5 kDa N/A N/A

Insulin A Chain 2.5 kDa N/A N/A

Continued on next page

Calibrating Protein Molecular Weight, Continued

Assigned Apparent Molecular Weights, continued

SeeBlue

Pre-Stained

Standard

NuPAGE

(4–12%)

Bis-Tris/MES

NuPAGE

(4–12%)

Bis-Tris/MOPS

NuPAGE

(3–8%)

Tris-Acetate

Myosin 188 kDa 191 kDa 210 kDa

BSA 62 kDa 64 kDa 71 kDa

Glutamic Dehydrogenase 49 kDa 51 kDa 55 kDa

Alcohol Dehydrogenase 38 kDa 39 kDa 41 kDa

Carbonic Anhydrase 28 kDa 28 kDa N/A

Myoglobin 18 kDa 19 kDa N/A

Lysozyme 14 kDa 14 kDa N/A

Aprotinin 6 kDa N/A N/A

Insulin 3 kDa N/A N/A

SeeBlue

Plus2 Pre-

Stained Standard

NuPAGE

(4–12%)

Bis-Tris/MES

NuPAGE

(4–12%)

Bis-Tris/MOPS

NuPAGE

(3–8%)

Tris-Acetate

Myosin 188 kDa 191 kDa 210 kDa

Phosphorylase B 98 kDa 97 kDa 111 kDa

BSA 62 kDa 64 kDa 71 kDa

Glutamic Dehydrogenase 49 kDa 51 kDa 55 kDa

Alcohol Dehydrogenase 38 kDa 39 kDa 41 kDa

Carbonic Anhydrase 28 kDa 28 kDa N/A

Myoglobin 17 kDa 19 kDa N/A

Lysozyme 14 kDa 14 kDa N/A

Aprotinin 6 kDa N/A N/A

Insulin 3 kDa N/A N/A

Troubleshooting

Problem Cause Solution

Run taking longer

time

Running buffer too dilute Make fresh running buffer as described on

page 15 and do not adjust the pH of the 1X

running buffer.

Low or no current

during the run

Incomplete circuit

 Remove the tape from the bottom of the

cassette prior to electrophoresis.

 Make sure the buffer covers the sample

wells.

 Check the wire connections on the buffer

core to make sure the connections are intact.

Streaking of proteins

 Sample overload

 High salt concentration in

the sample

 Sample precipitates

 Contaminants such as

membranes or DNA

complexes in the sample

 Load the appropriate amount of protein as

described on page 10.

 Decrease the salt concentration of your

sam

ple using dialysis or gel filtration.

 Increase the concentration of SDS in your

sample, if necessary to maintain the

solubility of the protein.

 Centrifuge or clarify your sample to remove

particulate contaminants.

Dumbbell shaped

bands after

electrophoresis

Loading a large volume of

sample causes incomplete

stacking of the entire sample.

This effect is more intensified

for larger proteins.

Load the appropriate volume of sample per

well as described on page 10. If your sample is

too dilute, concentra

te the sample using

ultrafiltration.

Continued on next page

Troubleshooting, Continued

Using incorrect

Buffers with

NuPAGE

Bis-Tris

Gels

See the table below for the outcome of your results if you accidentally used an

incorrect buffer instead of the NuPAGE

MOPS/MES SDS Running Buffer and

NuPAGE

LDS Sample Buffer on the NuPAGE

Bis-Tris Gels.

If you used the… Instead of the…. Then….

the run time of the gel is decreased by

~10–15 minutes.

NuPAGE

MES SDS

Running Buffer

NuPAGE

MOPS SDS

Running Buffer

there is decreased separation and

resolution for proteins >36 kDa.

the run time of the gel is increased by

~10–15 minutes.

NuPAGE

MOPS SDS

Running Buffer

NuPAGE

MES SDS

Running Buffer

the lower molecular weight proteins

(<14 kDa), which are normally well

resolved, are not resolved while the high

molecular weight proteins are resolved

more than normal.

Novex

Tris-Glycine

SDS Sample Buffer

NuPAGE

LDS Sample

Buffer

some bands are not very sharp and there

is increased protein fragmentation.

Novex

Tricine SDS

Sample Buffer

NuPAGE

LDS Sample

Buffer

the band sharpness is not affected, but

the lanes will be slightly wider due to the

increased amount of SDS and buffer salts

from the Tricine Sample Buffer.

the gel will have an extremely long run

time of 3–4 hours due to the low

migration of the glycine ions at neutral

pH.

the sensitivity of the staining for high

molecular weight proteins is decreased.

Novex

Tris-Glycine

SDS Running Buffer

and the Novex

Tris-

Glycine SDS Sample

Buffer

NuPAGE

MOPS or

MES SDS Running

Buffer and the

NuPAGE

LDS Sample

Buffer

the bands are more compressed at the

bottom of the gel, regardless of the gel

percentage and the bands have a cupped

appearance at the bottom of the band.

the run time of the gel is increased by

1–2 hours due to the slow migration of

the tricine ions at neutral pH.

Novex

Tricine SDS

Running Buffer and the

Tricine SDS Sample

Buffer

NuPAGE

MOPS or

MES SDS Running

Buffer and the

NuPAGE

LDS Sample

Buffer

there may be background streaking in

the lanes.

Continued on next page

Troubleshooting, Continued

Using incorrect

Buffers with

NuPAGE

Tris-

Acetate Gels

Refer to the table below for the outcome of your results if you accidentally used

an incorrect buffer system instead of the NuPAGE

Tris-Acetate SDS Running

Buffer and NuPAGE

LDS Sample Buffer on the NuPAGE

Tris-Acetate Gels.

Sample Buffer Running Buffer Antioxidant

Results

Novex

Tris-Glycine

SDS

NuPAGE

Tris-Acetate SDS Yes Fuzzy, smeared bands.

Novex

Tricine SDS NuPAGE

Tris-Acetate SDS Yes Bands are not very sharp.

NuPAGE

LDS NuPAGE

MES SDS or

NuPAGE

MOPS SDS

Yes Bands are diffuse and have a

“U” shape. More low

molecular weight proteins are

visible.

NuPAGE

LDS Novex

Tris-Glycine SDS No The run time is twice as long

as the Tris-Acetate Buffer

system. The band resolution is

poor.

NuPAGE

LDS Novex

Tricine SDS No The run time is 10–15 minutes

faster than the Tris-Acetate

Buffer system. Reduced

protein bands are diffuse

while non-reduced large

molecular weight protein

bands are smeared.

Novex

Tris-Glycine

SDS

Novex

Tris-Glycine SDS No The run time is much longer

than the Tris-Acetate Buffer

system and the bands are very

faint with a streaked

background. Fewer low

molecular weight bands are

resolved.

Novex

Tricine SDS Novex

Tricine SDS No The run time is 10–15 minutes

faster than the Tris-Acetate

Buffer system and reduced

protein bands are not sharp.

The overall performance is

acceptable.

Appendix

Accessory Products

Electrophoresis

Reagents

A large variety of electrophoresis reagents and apparatus are available from

Invitrogen for the separation and analysis of proteins. Ordering information is

provided below. For more information, visit our website at

www.invitrogen.com or call Technical Support (see page 56).

Product Quantity Catalog no.

XCell

SureLock

™

Mini-Cell 1 unit EI0001

XCell II

™

Blot Module 1 unit EI9051

PowerEase

500 Power Supply 1 unit EI8600

DryEase

Mini-Gel Drying System 1 Kit NI2387

StainEase

Staining Tray 2/pack NI2400

Gel-Dry

™

Drying Solution 500 mL LC4025

iBlot

Gel Transfer Device 1 unit IB1001

Novex

Semi-Dry Blotter 1 unit SD1000

NuPAGE

LDS Sample Buffer (4X) 10 mL

250 mL

NP0007

NP0008

NuPAGE

MOPS SDS Running Buffer (20X) 500 mL NP0001

NuPAGE

MES SDS Running Buffer (20X) 500 mL NP0002

NuPAGE

Tris-Acetate SDS Running Buffer (20X) 500 mL LA0041

NuPAGE

Antioxidant 15 mL NP0005

NuPAGE

Sample Reducing Agent (10X) 250 μL

10 mL

NP0004

NP0009

NuPAGE

Transfer Buffer (20X) 125 mL NP0006

Novex

Tris-Glycine Native Running Buffer (10X) 500 mL LC2672

Novex

Tris-Glycine Native Sample Buffer (2X) 20 mL LC2673

Nitrocellulose Membrane 0.2 μm 20 membrane/filter papers LC2000

Invitrolon

™

PVDF membranes 0.45 μm 20 membrane/filter papers LC2005

PVDF membranes 0.2 μm 20 membrane/filter papers LC2002

Continued on next page

Accessory Products, Continued

Protein Standards

and Stains

Ordering information for stains and protein molecular weights is provided

below. For more information, visit our website at www.invitrogen.com or

contact Technical Support (see page 56).

Product Application Quantity Catalog no.

SimplyBlue

™

Safe-Stain

Fast, sensitive, safe Coomassie G-250

staining of proteins in polyacrylamide gels

1 L LC6060

SilverQuest

™

Silver Staining

Kit

Sensitive silver staining of proteins

compatible with mass spectrometry

analysis

1 Kit LC6070

Colloidal Blue Staining Kit

Sensitive colloidal Coomassie G-250

staining of proteins in polyacrylamide gels

1 Kit LC6025

SilverXpress

Silver

Staining Kit

High-sensitivity, low background protein

and nucleic acid silver staining

1 Kit LC6100

Mark 12

™

Unstained

Standard

For estimating the apparent protein

molecular weight

1 mL LC5677

MagicMark

™

Western

Standard

For protein molecular weight estimation on

western blots

250 μL LC5600

SeeBlue

Pre-Stained

Standard

For monitoring the progress of your run

and evaluating transfer efficiency

500 μL LC5625

SeeBlue

Plus2 Pre-Stained

Standard

For visualizing protein molecular weight

range and evaluating transfer efficiency

500 μL LC5925

Novex

Sharp Pre-stained

Protein Standard

For visualizing protein molecular weight

range and evaluating transfer efficiency

2 × 250 μL LC5800

BenchMark

™

Protein

Ladder

For estimating the apparent protein

molecular weight

2 × 250 μL 10747-012

Recipes

NuPAGE

MOPS

SDS Running

Buffer

The NuPAGE

MOPS SDS Running Buffer (20X) is available from Invitrogen (see

page 48).

50 m

M MOPS

50 mM Tris Base

0.1% SDS

1 mM EDTA

pH 7.7

1. To prepare 500 mL of 20X NuPAGE

MOPS SDS Running Buffer, dissolve the

following reagents to 400 mL ultrapure water:

MOPS 104.6 g

Tris Base 60.6 g

SDS 10 g

EDTA 3.0 g

2. Mix well and adjust the volume to 500 mL with ultrapure water.

3. Store at +4C. The buffer is stable for 6 months when stored at +4C.

4. For electrophoresis, dilute this buffer to 1X with water (see page 15). The pH of

the 1X solution is 7.7. Do not use acid or base to adjust the pH.

NuPAGE

MES

SDS Running

Buffer

The NuPAGE

MES SDS Running Buffer (20X) is available from Invitrogen (see

page 48).

50 m

M MES

50 mM Tris Base

0.1% SDS

1 mM EDTA

pH 7.3

1. To prepare 500 mL of 20X NuPAGE

MES SDS Running Buffer, dissolve the

following reagents to 400 mL ultrapure water:

MES 97.6 g

Tris Base 60.6 g

SDS 10 g

EDTA 3.0 g

2. Mix well and adjust the volume to 500 mL with ultrapure water.

3. Store at +4C. The buffer is stable for 6 months when stored at +4C.

4. For electrophoresis, dilute this buffer to 1X with water (see page 15). The pH of

the 1X solution is 7.3. Do not use acid or base to adjust the pH.

ontinued on next page

Recipes, Continued

NuPAGE

Tris-

Acetate SDS

Running Buffer

The NuPAGE

Tris-Acetate SDS Running Buffer (20X) is available from Invitrogen

(see page 48).

50 m

M Tricine

50 mM Tris Base

0.1% SDS

pH 8.24

1. To prepare 500 mL of 20X NuPAGE

Tris-Acetate SDS Running Buffer, dissolve

the following reagents to 400 mL ultrapure water:

Tricine 89.5 g

Tris Base 60.6 g

SDS 10 g

2. Mix well and adjust the volume to 500 mL with ultrapure water.

3. Store at +4C. The buffer is stable for 6 months when stored at +4C.

4. For electrophoresis, dilute this buffer to 1X with water (see page 15). The pH of

the 1X solution is 8.24. Do not use acid or base to adjust the pH.

Tris-Glycine

Native Running

Buffer

The Tris-Glycine Native Running Buffer is available from Invitrogen (see page 48).

25 mM Tris Base

192 mM Glycine

pH 8.3

1. To prepare 1,000 mL of 10X Tris-Glycine Native Running Buffer, dissolve the

following reagents to 900 mL ultrapure water:

Tris Base 29 g

Glycine 144 g

2. Mix well and adjust the volume to 1,000 mL with ultrapure water.

3. Store at room temperature. The buffer is stable for 6 months when stored at

room temperature.

4. For native electrophoresis, dilute this buffer to 1X with water (see page 51). The

pH of the 1X solution is 8.3. Do not use acid or base to adjust the pH.

ontinued on next page

Recipes, Continued

NuPAGE

LDS

Sample Buffer

The NuPAGE

LDS Sample Buffer (4X) is available from Invitrogen (see page 48).

106 mM Tris HCl

141 mM Tris Base

2% LDS

10% Glycerol

0.51 mM EDTA

0.22 mM SERVA Blue G250

0.175 mM Phenol Red

pH 8.5

1. To prepare 10 mL of 4X NuPAGE

LDS Sample Buffer, dissolve the following

reagents to 8 mL ultrapure water:

Tris HCl 0.666 g

Tris Base 0.682 g

LDS 0.800 g

EDTA 0.006 g

Glycerol 4 g

SERVA Blue G250 (1% solution) 0.75 mL

Phenol Red (1% solution) 0.25 mL

2. Mix well and adjust the volume to 10 mL with ultrapure water.

3. Store at +4C. The buffer is stable for 6 months when stored at +4C.

4. For electrophoresis, prepare your samples in this buffer as described on page 14.

The pH of the 1X solution is 8.5. Do not use acid or base to adjust the pH.

Tris-Glycine

Native Sample

Buffer

The Tris-Glycine Native Sample Buffer is available from Invitrogen (see page 48).

1X composition

100 mM Tris HCl

10% Glycerol

0.0025% Bromophenol Blue

pH 8.6

1. To prepare 10 mL of 2X Tris-Glycine Native Sample Buffer, mix the following

reagents :

0.5 M Tris HCl, pH 8.6 4 mL

Glycerol 2 mL

0.1% (w/v) Bromophenol Blue 0.5 mL

2. Adjust the volume to 10 mL with ultrapure water.

3. Store at +4C. The buffer is stable for 6 months when stored at +4C.

4. Use this buffer to prepare samples for non-denaturing NuPAGE

Tris-Acetate

gel electrophoresis (see page 52).

ontinued on next page

Recipes, Continued

NuPAGE

Transfer

Buffer

The NuPAGE

Transfer Buffer (20X) is available from Invitrogen (see page 48).

25 mM Bicine

25 mM Bis-Tris (free base)

1 mM EDTA

pH 7.2

1. To prepare 125 mL of 20X NuPAGE

Transfer Buffer, dissolve the following

reagents to 100 mL ultrapure water:

Bicine 10.2 g

Bis-Tris (free base) 13.1 g

EDTA 0.75 g

2. Mix well and adjust the volume to 125 mL with ultrapure water.

3. Store at +4C. The buffer is stable for 6 months when stored at +4C.

4. For western transfer, dilute this buffer to 1X with water (see page 35). The pH

of the 1X solution is 7.2. Do not use acid or base to adjust the pH.

Gel Migration Chart

NuPAGE

Bis-Tris

Gel Migration

Chart

The migration patterns of protein standards* on NuPAGE

Bis-Tris and Tris-

Acetate Gels are shown on the table below. Use the table to select the proper gel for

separating proteins based on size. Optimal resolution is achieved when protein

bands migrate within the shaded regions.

55 kDa

40 kDa

290 kDa

240 kDa

160 kDa

116 kDa

160 kDa

97 kDa

66 kDa

40 kDa

500 kDa

290 kDa

500 kDa

% of length of gel

10%

Bis-Tris Gel

w/ MES

Running

Buer

10%

Bis-Tris Gel

w/ MOPS

Running

Buer

4-12%

Bis-Tris Gel

w/ MES

Running

Buer

4-12%

Bis-Tris Gel

w/ MOPS

Running

Buer

200 kDa

116 kDa

97 kDa

66 kDa

36 kDa

31 kDa

21 kDa

14 kDa

6 kDa

3.5 kDa

2.5 kDa

55 kDa

100

14 kDa

116 kDa

97 kDa

3-8%

TA Gel

w/ TA

Running

Buer

TA Gel

w/ TA

Running

Buer

12%

Bis-Tris Gel

w/ MES

Running

Buer

12%

Bis-Tris Gel

w/ MOPS

Running

Buer

200 kDa

116 kDa

97 kDa

66 kDa

36 kDa

31 kDa

21 kDa

14 kDa

6 kDa

3.5 kDa

2.5 kDa

55 kDa

14 kDa

On NuPAGE

Bis-Tris Gels, bands correspond to the migration of Mark12™

Unstained Standard (Cat. no. LC5677) under denaturing conditions; on NuPAGE

Tris-Acetate Gels, bands correspond to the migration of HiMark™ Unstained

Standard (LC5688) under denaturing conditions.

6 kDa

66 kDa

etatecA-sirTsirT-siB

Corresponding

Tris- Glycine

Gel:

Corresponding

Tris- Glycine

Gels:

4% or 6%

55 kDa

Gel Conversion Chart

Tris-Glycine/

Tricine to

NuPAGE

Gel

Conversion Chart

Use the following table for determining the appropriate NuPAGE

Bis-Tris or Tris-

Acetate Gel that is recommended for a similar Tris-Glycine or Tricine gel.

Currently using: Recommended NuPAGE

Gel

4% Tris-Glycine 3-8% NuPAGE

Tris-Acetate (+ TA Buer)*

6% Tris-Glycine 3-8% NuPAGE

Tris-Acetate (+ TA Buer)

8% Tris-Glycine 7% NuPAGE

Tris-Acetate (+ TA Buer)

10% Tris-Glycine 10% NuPAGE

Bis-Tris (+ MOPS Buer)

12% Tris-Glycine 10 % NuPAGE

Bis-Tris (+ MOPS Buer)

14% Tris-Glycine 12% NuPAGE

Bis-Tris (+ MOPS Buer)

16% Tris-Glycine 12% NuPAGE

Bis-Tris (+ MES Buer)

18% Tris-Glycine 12% NuPAGE

Bis-Tris (+ MES Buer)

4-12% Tris-Glycine 3-8% NuPAGE

Tris Acetate (+ TA Buer) or

4-12% NuPAGE

Bis-Tris (+ MOPS Buer)

4-20% Tris-Glycine 4-12% NuPAGE

Bis-Tris (+ MES Buer)

8-16% Tris-Glycine 4-12% NuPAGE

Bis-Tris (+ MOPS Buer)

10-20% Tris-Glycine 12% NuPAGE

Bis-Tris (+ MOPS Buer)

10% Tricine 10 % NuPAGE

Bis-Tris (+ MES Buer)

16% Tricine 4-12% NuPAGE

Bis-Tris (+ MES Buer) or

12% NuPAGE

Bis-Tris (+ MES Buer)

10-20% Tricine 4-12% NuPAGE

Bis-Tris (+ MES Buer)

* Resolution on a 3-8% NuPAGE

Novex Tris-Acetate gel is better than on a

4% Tris-Glycine gel, but the molecular weight separation range is not as wide.

See the migration chart at the left for migration patterns.

ent

y us

commen

Technical Support

World Wide Web

Visit the Invitrogen website at www.invitrogen.com for:

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ontinued on next page

Technical Support, Continued

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References

Kubo, K. (1995) Effect of Incubation of Solutions of Proteins Containing Dodecyl Sulfate on the Cleavage

of Peptide Bonds by Boiling. Anal Biochem. 225, 351-353.

Laemmli, U. K., (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage

T4. Nature, 227, 680-685.

Moos, M Jr., Nguyen, N. Y., Liu, T.Y. (1988) Reproducible High Yield Sequencing of Proteins

Electrophoretically Separated and Transferred to an Inert Support. J. Biol. Chem. 263, 6005-6008.

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