2D proteomics has become a method of choice for comparing the complete protein profile of any two cells (be it from the same or different organisms). Isoelectric point (pI) and molecular weight are used to separate these biomolecules in two dimensions (hence the term “2D”), thereby aiding analysis.
2D electrophoresis is performed in two steps. The first step is isoelectric focusing which separates proteins on the basis of their pI and the second step is normal SDS-PAGE in which proteins are separated on the basis of their molecular weight. Preparation of the sample for the first dimension is a critical step and the presence of impurities such as nucleic acids, lipids and salts, pose a major hindrance in effective separation of proteins in the 1st dimension. The 2D Clean-Up Kit from GE Healthcare (formerly Amersham Biosciences) is the most effective method of getting rid of such impurities and thus, sample preparation with this kit results in proper isoelectric focusing and minimal streaking.
The kit can be used in two cases: when the sample volume is 1-100 ul containing 1-100 ug of protein, and when both sample volume and protein amount of protein are greater than 100 ul and 100 ug, respectively. Only a slight modification of the protocol is required in the latter case, wherein an extra “wash additive” solution provided with the kit is used. The basic steps of the protocol are, otherwise, the same.
Two solutions, “precipitant” and “co-precipitant”, are provided with the kit and are used to precipitate proteins from a crude cell extract. Care should be taken to not disturb the pellet after centrifugation. Wash buffer is then used to wash the protein pellet obtained in the previous step. Wash buffer should be cooled to –20ºC prior to use. The pellet should be resuspended in the wash buffer properly to remove the contaminants completely. At this step, the samples can be stored at –20ºC for up to a week. Pure protein, free from any interfering agents, is obtained upon centrifuging the samples at this step. The pellet is then air dried for a very short period. Extensive drying renders the pellet difficult to resuspend in rehydration buffer (not provided with the kit). The chemicals required for the preparation of rehydration buffer should be of high quality.
I have used the kit for preparing samples from bacterial cells. The cells are lysed in a low salt buffer by sonication. I process the lysate using this kit to remove all the non-protein contaminants. The kit also helps in the removal of membrane proteins. Since I only look for cytoplasmic proteins, this is not a problem. I generally process 4-6 samples at one time. However, a greater number of samples could be processed at a time. After preparation, the samples are run on Ettan IPGphor IEF System (GE Healthcare). After separation in the first dimension, the strip is run on the second dimension and the gels are either commassie or silver stained.
The contents of the kit are sufficient for 50 purifications. The kit is stable for more than one year and the reagents can be stored at room temperature. However, the wash buffer should be kept at –20ºC for at least one hour prior to use. I have found the kit to be very effective in removing non-protein contaminants from the cell lysate. The entire procedure takes nearly one hour. The gel obtained using this protein sample has fewer streaks and much cleaner spots as compared with conventional sample prep procedures. The results are reproducible and I have observed that there is no loss in the intensity of the spots over multiple 2D gels with similar samples.
Indian Institute of Science
Molecular Biophysics Unit