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™.
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.
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.
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
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
The Mini-PROTEAN® system includes the four-gel Mini-PROTEAN Tetra cell 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 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
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
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.
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.
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