Bio-Rad Criterion XT Tris-Acetate Precast Gels User Manual

Criterion XT™Precast Gel

Instruction Guide

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Table of Contents

Section 1 General Information.............................................................................................. 1

1.1 Introduction........................................................................................................................................... 1

1.2 Criterion XT Precast Gels...................................................................................................................... 2

1.3 Criterion System Specifications............................................................................................................. 4

1.4 Criterion XT Comb Configurations......................................................................................................... 4

Section 2 Setup and Basic Operation................................................................................... 5

2.1 Setting Up and Running Criterion XT Gels.............................................................................................5

2.2 Opening Criterion XT Cassettes and Removing the Gels...................................................................... 6

Section 3 SDS-PAGE and Native PAGE................................................................................. 7

3.1 Criterion XT Gel Selection Guide........................................................................................................... 7

3.2 Bis-Tris Gel Composition...................................................................................................................... 9

3.3 Tris-Acetate Gel Composition............................................................................................................... 9

3.4 Criterion XT Buffers and Reagents........................................................................................................ 9

3.5 Sample Preparation..............................................................................................................................10

3.6 Running Conditions .............................................................................................................................10

Section 4 2-D Electrophoresis............................................................................................ 11

4.1 Equilibration....................................................................................................................................... 11

4.2 Agarose Overlay ............................................................................................................................... 11

Section 5 Staining and Detection....................................................................................12–13

5.1 SDS-PAGE and Native PAGE Detection.........................................................................................12–13

Section 6 Blotting................................................................................................................ 14

Section 7 Troubleshooting..............................................................................................15–16

Section 8 Ordering Information......................................................................................17–22

8.1 Criterion XT Gels................................................................................................................................ 17

8.2 Criterion XT Buffers and Kits ............................................................................................................. 17

8.3 Other Related Products..................................................................................................................... 17

8.4 Criterion Gels.................................................................................................................................18–19

8.5 Criterion Gel Accessories................................................................................................................... 20

8.6 Protein Standards ............................................................................................................................. 20

8.7 Detection Reagents........................................................................................................................... 21

8.8 Blotting Membranes.......................................................................................................................... 22

8.9 Equipment......................................................................................................................................... 22

Section 1
General Information

1.1 Introduction

Criterion is the next generation of dedicated precast gel systems. The innovative, easy-to-use design
produces superior resolution while allowing you to run more samples per gel. Compared to any other precast
gel system, Criterion produces more results while providing significant cost and time savings. Some of the
unique features and benefits provided are:

• 12 month shelf life for Bis-Tris gels

• 8 month shelf life for Tris-acetate gels

• Room temperature storage for Bis-Tris gels

• Easy sample preparation without extra anti-oxidant addition steps

• Patented integral buffer chamber that eliminates buffer leaks

• Up to 26 sample capacity per gel

• Flexibility to run one or two gels

• Multichannel pipet compatible gels

• Outlined and numbered wells that simplify sample loading

• J-foot that improves gel drying and blotting results

US Patents #5,073,246, #5,656,145, #6,093,301 and other patents issued and pending.

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1.2 Criterion XT Precast Gels

Criterion XT precast gels are formulated at pH near neutrality to optimize gel matrix stability, significantly
delaying acrylamide hydrolysis, which occurs in traditional Laemmli systems. Specially optimized buffers
result in tight, consistently resolved bands throughout the life of the gel.

This versatile system allows the separation of small to large proteins using just two gel buffer systems:
Criterion XT Bis-Tris precast gels for small to mid-sized proteins and Criterion XT Tris-acetate precast gels for
large proteins.

The Criterion XT Bis-Tris gels are based on a Bis-Tris.HCl buffer system (pH 6.4) that uses discontinuous
chloride and MES or MOPS ion fronts to form moving boundaries to stack and then separate denatured
proteins by size. The chemistry of the XT Bis-Tris gels allows maximum stability and consistent results for a
minimum of one year. Running the same XT Bis-Tris gels with the XT MES denaturing running buffer or the XT
MOPS denaturing running buffer will produce different migration patterns. A combination of these two running
buffers and our three XT Bis-Tris gels can produce up to six different migration patterns in the small and mid­size range.

The Criterion XT Tris-acetate gels are based on a Tris-acetate buffer system (pH 7.0). It uses discontinuous
acetate and Tricine ion fronts to form moving boundaries to stack and then separate large denatured proteins
by molecular weight. The Criterion XT Tris-acetate gels can also be used to separate proteins by their charge­to-mass ratio (under native-PAGE conditions). This is possible because the XT Tris-acetate gels are made
without SDS, allowing the sample buffer and running buffer to dictate the separation mechanism. The
nonreducing and nondenaturing environment of native PAGE allows the detection of biological activity and
can improve antibody detection. Native PAGE can also be used to resolve multi-protein bands where
molecular mass separation by SDS-PAGE would reveal only one and for the separation of intact protein

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complexes. Separation by native PAGE with XT Tris-acetate gels uses discontinuous acetate and glycine
ion fronts to form moving boundaries to stack and separate proteins by both size and charge.

Protein samples for the Criterion XT precast gel system are prepared in a reducing denaturing sample buffer.
The sample buffer contains XT reducing agent, a pH neutralized and stabilized solution of TCEP as the
reducing agent; heat and SDS are used to denature the proteins. In addition, the use of TCEP in combination
with Bio-Rad’s optimized running buffers maintains proteins in a fully reduced state during the electrophoresis
run, eliminating the need for an anti-oxidant in the upper buffer chamber. Criterion XT Tris-acetate precast
gels can also be used for native PAGE. Proteins are prepared in a nonreducing, nondenaturing sample buffer,
which maintains the proteins’ native structure and charge density.

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1.3 Criterion System Specifications

Gel material Polyacrylamide

Gel dimensions (W x L) 13.3 x 8.7 cm

Gel thickness 1.0 mm

Resolving gel height 6.5 cm

Cassette dimensions (W x L) 15.0 x 10.6 cm

Cassette material Styrene copolymer

Comb material Polycarbonate

Storage tray material PET

Upper running buffer volume 60 ml

Lower running buffer volume 800 ml

Storage conditions Bis-Tris gels: Store flat at ambient temperature; DO NOT FREEZE

Tris-acetate gels: Store flat at 4°C; DO NOT FREEZE

Gel shelf life 12 months for Bis-Tris gels; 8 months for Tris-acetate gels

1.4 Criterion XT Comb Configurations

Comb Load Volume Comments

12+2 well 45 µl with two 15 µl reference wells Multichannel pipet compatible
18-well 30 µl
26-well 15 µl Multichannel pipet compatible
Prep+2 well 800 µl with two 15 µl reference wells
IPG 11 cm ReadyStrip
IPG+1 well 11 cm ReadyStrip IPG strip with one 15 µl reference well



IPG strip

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Section 2
Setup and Basic Operation

2.1 Setting Up and Running Criterion XT Gels

1. Each Criterion XT gel is packaged individually in a plastic storage tray. Remove the cover by gently pulling
the corner tab up and diagonally across the package. Remove the gel from the package.

2. Remove the comb and gently rinse the wells with ddH

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O or running buffer.

3. Remove the tape from the bottom of the cassette by pulling the

tab across the gel.

4. Insert the Criterion XT gel into one of the slots in the Criterion cell
tank. Ensure that each integral buffer chamber faces the center of
the cell.

5. Fill each integral buffer chamber with 60 ml running buffer.

6. Load samples using a Hamilton syringe or a pipet with gel loading
tips. A sample loading guide can be placed on the outer edge of
the cassette to aid in aligning pipet tips with the wells. This is
especially useful with multichannel pipets.

7. Fill each half of the lower buffer tank with 400 ml of running buffer

to the marked fill line.

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