I collected whole cell lysates of HT29 Clones in RIPA buffer (Protease Inhibitor cocktail, PMSF, Na3VO4 and NaF all added into the RIPA buffer) and analyzed them by western blot on the same day and saw expression of my protein of interest.
However, when I used the same sample to run another western blot a week later, the protein does not seem to be expressed. Can proteins degrade when kept in RIPA buffer over a period of time, in such a way that the expression can no longer be detected? I stored the lysates at -20C the entire time.
Well, we store our RIPA lysates in -80C and we have no problem with repeated measures. So, -20 might be the first problem. But still, western blotting consists of many steps and therefore is prone to errors. I'd suggest checking the protein content by total protein quantification and then I'd check something like coomassie or silver staining of gels instead of blotting to verify, whether the proteins look normally on gels.
I guess you mean protein degradation.
If so, the answer is yes and several factors could account for the degradation of proteins. First, you should check if your protease inhibitors are working properly, then your specific protein might have a very low stability.
I had the same problem with brain lysate and what I did was to make aliquotes of the sample to avoid repetitive thaw/freeze cycles, storage at -80°C and I don't wait too long to do all the experiments I need with the sample.
I hope that helps.
Good luck!
As Gonzalo mentioned, yes for sure you can have degradation of proteins. Was your loading control degraded as well? If so, that should indicate that your protease inhibitor is not working or you are not using enough. Also, PMSF only inhibits serine proteases, I think. You may want to add other inhibitors or EDTA or even SDS (will denature/inactivate many enzymes). The Roche complete protease inhibitors are pretty great too. Some proteins are also quite sensitive to freeze/thaw as well. For instance, it is not recommended to freeze your lysates when studying the autophagy marker LC3.
Good luck.
Thanks a lot for the replies. I will try storing the proteins in -80 instead of -20 and avoid repeated freezing and thawing of the samples.
My loading control was fine. No degradation products at all. I guess this would mean that my PMSF and Protease Inhibitors are fine.
Some loading controls (like actin or beta-tubulin) are abundant and slight degradation may not be detected. So, you may get fooled into thinking the protease inhibitors work. If you have a relatively minor protein in your lysate that always shows up--it may be an additional control that helps-because if its degraded-it will disappear!
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