I trypsinized the cells, incubated 5 min. washed with media and spun down, all at RT, as if to split them. After spinning, however, I just left them as a pellet in media while I finished my tissue culturing. After that, I aspirated the media and resuspended in ice cold PBS, centrifuged at 4 degree's, aspirated PBS, resuspended again to transfer cells to eppindorf tubes (all on ice). Centrifuged again at 4 degrees, aspirated PBS and flash froze in liquid N2, then transferred to -80 for use the next day. I'm afraid the 30 minutes they sat in media at room temp may have caused protein degradation, especially since I did not use protease or phosphitase inhibitors a this step. Any advice or tips would be appreciated.
Hi!
Probably, you are right. Try to use any protease inhibitor which is the most applicable for your work and do all manipulation on ice or (some times it is allowed in fridge (+4). If your centrifuge doesn't support +4 you can do short spin in maximally cooled tubes or longterm one in cold room.
You might actually be OK as 30 minutes is a relatively short period of time and, more importantly, you had not already lysed the cells. Since the cells were tightly packed in a pellet the proteins may be OK. What I would do is repeat the experiment and take the processing step through lysis of the cell pellet in the presence of a protease inhibitor cocktail.
Do your Western immunoblot analysis on the old sample and the new and see if there is a difference in either the banding pattern or intensity of the bands. If they are identical then you now have an n =2 for your experiment.
I think the protein will degradation. When you prepared to deal with the cell sample, make sure that every agent need pre-cool in 4 degrees. And all the step must be on the ice. Someone even did the sample collecting in the cold room.
You stored the cell sample in -80 degrees for use. However, I suggested if you can obtain the protein sample immediately, it is better to store the protein sample.
I think as long as the cell lysis process did not start you do not need to worry about protein degradation. However, I prefer once you trypsinize the cells, you start doing every step at 4 degrees. So, you can use ice cold media or PBS to re-suspend the cells and do the centrifugation at 4 degrees. Also try to minimize the time of waiting because even if the proteins will not be degraded, the cell signaling possibly could be affected by this waiting. The importance of this point would depend on the details of what your are testing.
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