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.