I've been performing protein extractions/Bradford assays no problem. Then the other day one of my cell pellets would not dissolve in RIPA/Halt protease inhibitor, leading to extremely low concentrations. It appears as a small, stringy glob at the bottom of my centrifuge tube that won't break up. There were approximately 4M cells and I had shown before that 200 uL of RIPA was sufficient.
The lysate had been stored in about 500 uL of RNALater, no PBS, then stored directly in the freezer. I just found out there is a better protocol for storage in RNALAter, so I will be using that from now on. However, I had previously used the exact same cell lysate and RIPA buffer a few weeks ago and everything worked fine. Is there any way to break up this pellet or any ideas as to why it isn't dissociating all of the sudden?
The goopy, stringy stuff you're talking about is probably all of the genomic DNA you released during lysis. You could try triturating using a 25-gauge needle to break that up, but be very careful not to foam up the solution. If you have access to a sonicator, that could help break everything up as well and would definitely lyse any remaining intact cells within your pellet. I've had great success using sonication on cartilage micromass cultures, which are very challenging to break up. In my experience 200uL is a pretty low volume for 4 million cells, though I've only scraped cells off the culture dish in RIPA, and not pelleted first. You can also just try increasing your RIPA volume to see if this helps. No good guesses as to why it worked before and not now though. Hope this helps!
