Second-Dimension Separation

The second-dimension of 2-D gel electrophoresis is a standard polyacrylamide gel. The proteins resolved by IEF on IPG strips in the first dimension are applied to an SDS polyacrylamide gel and separated by size (molecular weight) in a direction perpendicular to the first dimension.


This section provides details on polyacrylamide gel electrophoresis (PAGE) as part of the 2-D electrophoresis workflow. It discusses the different options available for the second-dimension electrophoresis and provides tips on how to transition from first to second dimension including IPG equilibration, IPG strip loading, and loading of standard plugs.


Related Topics: First-Dimension Separation (Isoelectric Focusing), Protein Sample Preparation for 2-D Electrophoresis, Staining and Visualization of Proteins After 2-D Electrophoresis, Imaging and Analysis of 2-D Electrophoresis Gels, Protein Spot Excision and Protein Identification, and Troubleshooting 2-D Electrophoresis Gels with 2-D Doctor™.

Page Contents
Second-Dimension Separation of Proteins

The pores of the second-dimension gel sieve proteins according to size because SDS coats all proteins with a negative charge essentially in proportion to their mass. The net effect is that proteins migrate as ellipsoids with a uniform negative charge-to-mass ratio, with mobility related logarithmically to mass. See figure below.

Schematic diagram showing separation of protiens by SDS-PAGE after separation by IEF.

Gel Composition

Homogeneous (single-percentage acrylamide) gels generally give excellent resolution of sample proteins that fall within a narrow MW range. Gradient gels have 2 advantages: they allow proteins with a wide range of MW to be analyzed simultaneously, and the decreasing pore size along the gradient functions to sharpen the spots.

Polyacrylamide gel is the most commonly used medium for protein electrophoresis; ready-to-use precast gels can be purchased, or you can hand cast polyacrylamide gels on the bench.

Second Dimension and High Throughput

Since the first dimension can be run in batches of 12 strips at a time, it is desirable to run the same number of samples in the second dimension. Precast gels ensure high reproducibility among samples and help reduce the work involved in running large numbers of samples. Alternatively, gels can be hand cast 12 at a time under identical conditions with multi-casting chambers.

Depending on the experiment, requirements for resolution, throughput, and speed may vary. Whereas resolution may be more important for analyzing complex samples, throughput is important when many samples must be run. Bio-Rad offers several lines of electrophoresis cells and precast gels for 2-D experiments.

System Selection Guide

First Dimension IEF Second-Dimension Electrophoresis
Focusing Cell IPG Length Vertical Electrophoresis Cell # Gels/Cell Gel Dimension Run Time

IEF system
18 or 24 cm
PROTEAN® Plus Dodeca™ cell
12 25 x 20.5 cm
20 x 20.5 cm

25 x 20.5 cm
6 hr
17 cm
2 18.3 x 19.3 cm 4–6 hr
17 cm
PROTEAN® II XL Multi-cell
6 18.3 x 20 cm
18.3 x 19.3 cm
4–6 hr
11 cm
Criterion™ cell
2 13.3 x 8.7 cm 30–60 min
11 cm
Criterion™ Dodeca™ cell
12 8.6 x 6.8 cm 60 min
7 cm
Mini-PROTEAN® Tetra cell
4 8.6 x 6.8 cm 15–45 min
7 cm
Mini-PROTEAN® 3 Dodeca™ cell
12 8.6 x 6.8 cm 35 min

Use this chart to find the second dimension system that best meets your needs based on IPG strip length, gel dimensions, and number of gels that can be run at once.

Mini Electrophoresis Systems

Mini Electrophoresis Systems

The Mini-PROTEAN Product Line includes the Mini-PROTEAN Tetra cell, which can hold up to four gels, and the high-throughput Mini-PROTEAN 3 Dodeca Cell (for running up to 12 mini gels). Mini gels can accommodate 7 cm IPG strips. Both handcast and Mini-PROTEAN TGX Precast Gels can be used with these cells.


Midi Electrophoresis Systems

Midi Electrophoresis Systems

Bio-Rad's Criterion™ midi gel system includes the Criterion™ cell (for 1–2 gels) and Criterion™ Dodeca™ cell (for 1–12 gels). Criterion precast gels provide increased resolution over a mini gel system but still run fast enough to let you generate 2-D data from your sample in a single day. They are available in a wide range of polyacrylamide percentages in both standard and extended shelf-life formulations.


Large Electrophoresis Systems

Large Electrophoresis Systems

For the highest resolution separations or to accommodate larger protein loads, choose from large format cells that are compatible with 17 cm (PROTEAN® II XL cell and PROTEAN® II XL multi-cell), or 18 or 24 cm IPG strips (PROTEAN® Plus Dodeca cell).

The PROTEAN II system provides the most flexibility with the ability to choose the glass plates, spacer, and sandwich clamps to cast two gel lengths: 16 or 20 cm. The PROTEAN Plus Dodeca cell allows maximum throughput with the capability to run up to 12 gels at a time. In general, whether your sample requires maximum flexibility or maximum throughput, these cells will meet your needs for reproducible, high-resolution results.

Options for PROTEAN II and PROTEAN Plus

Precast Gel Options Handcast Gel Options IPG Options Maximum # of Gels Run
XL Cell
No Yes 17cm 2
XL Multi Cell No Yes 17cm 6
PROTEAN Plus No Yes 18 and 24cm 12
PROTEAN II system can also be configured to accommodate 1-D applications


Transition from First to Second Dimension

The transition from 1-D to 2-D gel electrophoresis involves 2 steps: equilibration of the resolved IPG strips in SDS reducing and alkylating buffers using ReadyPrep 2-D starter kit equilibration buffer I and II, and embedding of the strip on the top of the second-dimension gel. Proper equilibration simultaneously ensures that proteins are coated with SDS and that cysteines are reduced and alkylated. This step is analogous to boiling samples in SDS buffer prior to SDS-PAGE. Rehydration/equilibration trays sized for each size strip can be used for equilibration. The equilibrated IPG strips are placed on top of the gel and fixed with molten agarose solution to ensure good contact between the gel and the strip.

Read complete directions for performing protein electrophoresis, including step-by-step protocols for IPG equilibration and the use of Precision Plus Protein™ standard plugs.


Products and Accessories
ReadyPrep™ 2-D Starter Kit
The ReadyPrep 2-D starter kit is intended for first-time users of the PROTEAN IEF cell and ReadyStrip IPG strips. The kit contains tested premixed reagents and a reference manual with technical tips.
PROTEAN® II xi Multi-Gel Casting Chamber
Multi-gel casting chamber, 20 x 20 cm gel size, for up to ten 1.5 mm thick gels. Sealing plate, gasket, separation sheets, acrylic blocks, and PROTEAN II XL alignment cards are included.
AnyGel™ Stand
Six-row gel stand, holds 6 PROTEAN gels, 12 Criterion gels, or 18 Ready Gel mini gels.
Premixed Buffers
Buffers and reagents are offered for each step of the 2-D separation process.
Precision Plus Protein™ Standard Plugs Precision Plus Protein™ Standard Plugs
Agarose plugs that are 1 mm thick and contain 10 Strep-tagged recombinant proteins (10—250 kD), including three reference bands.
Criterion Precast Gels Criterion Precast Gels
Criterion precast gels include a broad selection of mid-sized polyacrylamide gels in cassettes with integrated upper chambers.

Buffers, Reagents, and Other Tools

Several accessories are available to help with the second dimension run. These include: ReadyPrep 2-D starter kit, premixed buffers for gel casting, preparative electrophoresis tools such as Rotofor® and Mini Rotofor cells, multi-casting chambers, gradient formers, Precision Plus Protein standard plugs, AnyGel stands, and gel clips.


2-D Video Tutorial
How to run a 2-D Gel from start to finish.


Number Description Options
Protein Blotting Guide, Ver C
2-D Electrophoresis Workflow How-To Guide, Rev F


Number Description Options
Number Description Options
6222 IPG Equilibration for the Second Dimension Click to download
6236 Using Precision Plus Protein Standard Plugs Click to download