Which is the most efficient way to store samples for general western blotting (not phospho) for weeks, as cell pellet at -20°C or in RIPA/lysis buffer after lysis or protein with SDS sample buffer at -20°C or on the membrane or may be something else anyone can suggest? Thank you.
Hi Komal, I can recommend boiling the sample in SDS buffer for 5 minutes and storing them at -20 °C afterwards. Before running the SDS-PAGE, I thawed the samples by putting them at 95 °C for a couple of minutes. I still had good western blot results 3 or 4 months after sample preparation, never tested if the samples can be stored even longer. I hope that helps!
Add protease inhibitor cocktail to your lysis buffer. Store lysed samples in aliquots at -80C. Aliquots must be just enough for no of gels you run at a time.
I would also suggest to store the aliquots at -80°C: it may not be relevant for all the proteins, but for sure some of them may be degraded at -20°C. For example I have experience with a large leucine rich repeat kinase (LRRK2, which is involved in Parkinson's disease) and we observed that the protein disappeared by storing samples at -20°C even for a short period.
The question is: Are you probing for phospho markers?
If so, storage becomes much more important. I find that, like Edmund Pool, lysing in lysis buffer containing protease inhibitor and then storing frozen for short periods ie 2-3 weeks is fine even for many phospho proteins. Once thawed, lysates can be re-run for total protein but phospho protein staining will be variable!
You can also store blots on which protein has already been transferred. Blots, once dried, can be kept in sealed containers, sitting on paper tissue, in a refrigerator for long periods of time before probing with primary antibody.
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