I'm looking at phosphorylated proteins in triple negative breast cancer cells, so they are quite sensitive. I was wondering how long can I store my samples at 4°C after mixing them with loading dye and DTT and then boiling for 5mins?
Also (totally separate question), why do my cells clump together after adding RIPA lysis buffer? When I resuspend my cells in RIPA lysis buffer, they appear as a blob no matter how many times I resuspend. Will this affect western blot results, and do I overcome this problem?
Hello Maddie,
Sometimes, if I have leftover prepared samples, I freeze them again and run it whenever. I have never kept them at 4ºC. You don't need to boil again when running the rest of the sample.
I had never had my cells clump when adding RIPA lysis buffer, so I cannot help you with that. Do you directly lyse them after you retrieve the cells? Normally after lysing I centrifuge 15 mins at maximum speed to remove the membranes that are insoluble from the supernatant.
Hope it helps!
Irene
Hi Maddie Yeo ,
Like Irene Adán-Barrientos mentioned, I have never kept my samples at 4ºC after preparation. I would highly advise freezing the samples at -20ºC or lower for storage. Even after freezing the samples, I generally do a quick boil for about 2-3 minutes before loading the samples to the gel and the protein is quite stable this way.
As for the cell clumping, I had this issue with my membrane protein. It was fairly large (~250 kDa) and would tend to clump after adding RIPA lysis buffer. I had to do some trial and error with the volume of RIPA buffer added to the sample before I found the best ratio. Try to see if adding more RIPA buffer would help. Do you generally incubate your samples after adding RIPA for about 30-60 minutes at either RT or on ice? Doing this with frequently flicking of the samples to mix might help. After the incubation I used to spin the samples and collect the supernatant using a microcentrifuge (15 mins at ~14k RPM). I hope this helps!
Good luck,
Meera
As alluded to above, the clumping is likely insoluble membrane that you have to spin down before doing any downstream analyses. You may also get sticky strands of clear DNA if your cell density is high enough. Both of these will complicate concentration assays and western blots.
Meera J. Patel yes, I usually incubate them on ice for 15mins and then spin down. I already added 100uL of RIPA, but will try adding more the next time, thank you so much!
Irene Adán-Barrientos Ryan Hobson thanks for the advice!
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