The two-dimensional (2-D) gel is at the heart of proteomics. No other technique separates charge and size isomers of polypeptides as well as 2-D PAGE. Hundreds to thousands of polypeptides can be resolved in a single 2-D PAGE gel and every spot represents a pure or nearly pure polypeptide. These polypeptides can be quantified, tested for post-translational modifications, or extracted for mass spectrometry analysis. 2-D gels have provided much valuable information and they will continue to be an integral part of proteomics research for the foreseeable future.
The tools for 2-D PAGE are readily available and reasonably priced. Commercial manufacturers like Bio-Rad provide nearly every reagent and tool required for successful 2-D PAGE. While the actual running of 2-D PAGE is rather straightforward and easily learned, it can be quite challenging for first-time users. When getting started for the first time, it is important to focus on the technique rather than on the sample and reagent prep. The ReadyPrep 2-D Starter kit from Bio-Rad is not only designed as a single-use kit to facilitate first-time use of 2-D PAGE, but this set of 2-D PAGE reagents can also be used to validate the 2-D electrophoresis system.
The ReadyPrep 2-D Starter Kit provides all the necessary reagents, including the protein control, for a successful 2-D PAGE. The kit contains an E. coli protein sample, rehydration/sample buffer, equilibration buffer I and II, nanopure water, 30% glycerol solution, idoacetamide and ReadyPrep overlay agarose. The buffer formulations as well as the protocol have been optimized for the E. coli protein sample. Before beginning, the user must decide which length of IPG strips to use and how many samples to focus. The instructions provided with the kit describe the use of the E. coli protein sample with six pH 4-7 IPG strips. Two of these strips will be stained after the isoelectic focusing (IEF) to illustrate the successful completion of the IEF step. The remaining IPG strips will be carried through the second-dimension separation. The SDS-PAGE gels can be stained with Coomassie stain. The amount of sample applied to each gel is sufficient so that a large number of protein spots can be readily detected when using Coomassie stain. The 2-D gel maps for the E. coli protein are also included in the instruction manual for reference. The kit contains sufficient reagents for the separation of the E. coli protein sample on six 17 cm IPGs, ten 11 cm IPGs or sixteen 7 cm IPGs. The user has the flexibility to choose among three SDS-PAGE formats (7 cm IPG strips and Mini-PROTEAN II Ready Gel precast gels, 11 cm IPG strips and Criterion gels or 17 cm IPG strips and PROTEAN II Ready Gel precast gels).
The ReadyPrep 2-D Starter Kit is a single-use kit. After reconstitution of the lyophilized ReadyPrep rehydration/sample buffer, it cannot be reused due to the presence of DTT in the buffer. Only fresh DTT should be used for every 2-D PAGE. Other than that, the ReadyPrep 2-D Starter kit has proved to be handy and users can then proceed to separating their own samples with confidence. The plus point is that the E. coli protein sample can act as a positive control for the electrophoresis system as well.
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Benjamin Lau
Research Officer
Advanced Biotechnology and Breeding Center (ABBC)
Malaysian Palm Oil Board (MPOB)
Malaysia
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