ReadyPrep™ 2-D Cleanup Kit From Bio-Rad

In the lab, I use 2D electrophoresis for differential expression studies, where I compare plant samples from different stages of development to find differences in their protein expression. Plant proteins are difficult to extract and prepare for 2D electrophoresis because plant cells are rich in compounds that interfere with the 2D electrophoresis separation method. Among others, polyphenols can build irreversible complexes with proteins, while the polysaccharides and lipids can cause severe disturbances in the 2D gel. A common protocol used in my lab for extracting proteins from plant tissues involves grinding plant material in liquid nitrogen in low salt/Tris-HCl buffer containing protease inhibitor cocktail and denaturant. Although proteins extracted following this protocol produce good SDS-PAGE separations, they are not suitable for 2D electrophoresis. I have tested this protocol to prepare and separate proteins from plant tissues by 2D electrophoresis with little success.

Therefore, after obtaining the supernatant consisting of predominantly soluble proteins, I processed the sample using the ReadyPrep 2-D Cleanup Kit and I managed to get well-resolved proteins spots from 2D electrophoresis with minimal streaks. Without the kit, I had only managed to observe horizontal streaks at both ends of the 2D gel. In addition to that, the protein concentrations obtained with the ReadyPrep kit were relatively higher.

Basically, the ReadyPrep 2-D Cleanup Kit from Bio-Rad helps to remove substances which are known to interfere with IEF/2D electrophoresis from virtually any protein sample. Examples of these substances are ionic detergents, salts, lipids and plant-derived phenolic compounds. At the same time, the kit also concentrates proteins and thus, allows for loading larger amounts of protein, which can improve spot detection.

The kit can be used with 1 – 500 ìg of protein in 100 ìl of final volume. Scaling up is required if dilute samples or samples greater than 100 ìl need to be processed. The protocols are quite easy to follow with all the required reagents and the entire process can be completed in about one hour. After precipitation, the proteins are washed and then resuspended in the IEF/2D-compatible rehydration buffer of choice, ready for isoelectric focusing or Laemmli sample buffer, ready for SDS-PAGE. The kit provides sufficient reagents to purify 50 samples of 100 ìl.

The major drawback to the ReadyPrep Cleanup Kit is that the product is not very affordable. However, if there is no other protocol that can remove IEF/2D electrophoresis contaminants from the samples as efficiently as the kit, as in my case, I think it is well worth of it. In addition to that, I would think the kit should include the proteomic grade or other ultrapure water that is needed to wash the protein pellet. Overall, the ReadyPrep Cleanup Kit from Bio-Rad saves loads of time and gives tremendous results compared to other methods.