We're suddenly unable to get a good protein content after cell lysis preparation. I mean, almost no protein by coomasie blue staining of the gel, yet our protein standards/ladders show up fine. Previously, I had no problems with overall protein content or detecting desired bands with my protocols. We've purchased new RIPA buffer and protease inhibitors; varied lysis time, sonication, boiling, loading buffer...everything we can think of and none of it has made a difference. Does anyone have any ideas? Something I'm missing? Coinciding with the onset of this problem are other cellular changes that suggest our mice may be harboring a retrovirus. Is it possible that a viral protease is untouched by our inhibitor cocktail and be chewing up the proteins in our samples? I did try lysing with boiling laemmli buffer to overcome that possibility; it didn't help, but I may not have performed that correctly.
Any ideas or thoughts?
Hi Julie,
cell lysis is not effect by viruses. When you do lysis with lämmli buffer, lysis will work no matter what.
I would rather check your SDS Page buffers. Made any new buffers recently? Might be worth to make new buffers (especially Lämmli and running buffer, staining buffers). Run some positive controls on your gel (other cells, older samples from earlier experiments that worked.
Good luck
Best
Chris
Hi Julie,
I agree with Chris: make a fresh batch of running buffer, transfer buffer etc. Try first to run cell lysates for the cell lines that you have normally in culture in the lab, and check for actin or tubulin. That way you can check if you are having issues with the running buffer or the transfer. If you still experience issues, I would recommend you to buy pre-casted gels and ready to use buffers, to rule out any problems with your stocks.
I hope this helps, and good luck!
Ruth
...as with many questions, sufficient Information is missing. Do you know the number of cells you used for lysis ? We usually use proteins from 5x10exp4 cells per lane. Are you sure to have correctly diluted the lysibuffer you use? What means "boiling" . Really 100'C. Try to heaAT SAMPto t85 !VC to avoid micelle-Formation:
Hi Julie, although u did the sds Page perfectly because u could see the markers and ladders. Let us know what is the source , like solid tissue or mono layer cells .. did you quantify your proteins before loading ? I agree with Guenther sir, u need to have sufficient amount of cells .. have a lane for lysate with out viral infection as positive control ..
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