The vast majority of protein scientists will tell you that sample preparation can be one of the most difficult and critical aspects of performing 2D gel electrophoresis (2DGE). As a result, sample preparation protocols for 2DGE are worth taking a closer look at, as well as the evaluation of potential problems.
The most common aspects of sample preparation that can affect resolution during 2DGE are the concentration of salts in the sample and the presence of detergents. To remedy potential problems, we switched from detergent to mechanical lyses, however, we ran into many additional side effects, such as nuclear protein contamination, as a result. Our problems with detergent contamination were solved when we tried the Bio-Rad ReadyPrep 2-D Cleanup Kit.
The procedure, although time consuming, is not very difficult and can be mastered quickly. Only common lab equipment is necessary, including a centrifuge and a -20 C refrigerator. Some concerns raised in the actual procedure are that the commonly used tubes used for this experiment can react with acetone. So, the procedure suggests volume limitations to minimize this interaction. This can lead to problems with obtaining enough of a protein yield to run 2DGE.
The kit is based on the commonly used TCA precipitation procedure and provides contamination-free solutions. A disadvantage of the kit is cost, since TCA precipitation can be performed using few lab chemicals. In addition, at the termination of the procedure, samples are resuspended in a 2D sample buffer (not provided with kit) that does not permit concentration determinations using standard Bradford or Lowry assays. To address the incompatibility with these assays, Bio-Rad offers a specific Lowry assay (RC DC Protein Assay Kit) that is compatible with most 2D gel sample buffers.
Jeffrey William Perry
University of Michigan
Bio-Rad’s ReadyPrep 2-D Cleanup Kit
A fast reliable kit to prepare and improve a variety of samples for 2 D gel electrophoresis.
This kit is not a fix-all and problems may persist after use.
The Bottom Line
This kit is great at saving time and removing salts, detergents, lipids, and most phenolic compounds from protein samples, but it will not improve ALL samples.