Hi, i'm trying to prepare HaCaT cell lysate for biochemical assay studies. However, my protein concentration are too low (e.g 0.3mg/ml). My protocol is as follows:
1. Cultured HaCat in 6-wells plates (X2), seeding density of 4X10^6 cells/per well in DMEM media
2. Upon confluency of 80%, I washed with PBS, added treatment
3. Washed with ice-cold PBS
4. added 400ul RIPA lysis buffer per wlell (millipore, 20-188) and incubated 5 mins
5. scrapped the cells off the wells on ice
6. transferred to cold-microcentrifuge tube, incubate on ice with constant agitation for 30 mins
7. centrifuged at 12,000rom for 15 mins
8. transferred supernatant
yet my protein concentration is low, is there anything i'm doing wrong? I also read somewhere that mammalian cells does not require use of homogenizer/ sonicayor as the chemical lysis method is sufficient.
Edit: I forgot to mention that I did add Protease inhibitors to RIPA buffer (Thank you Malcolm Nobre for reminding me)
Hello Yogabaanu Ulaganathan
Yes, chemical lysis is sufficient. There is one point missing in your protocol.
Did you add protease inhibitor cocktail in the RIPA lysis buffer just before lysing the cells? RIPA lysis buffer from Millipore 20-188 does not seem to contain protease inhibitor cocktail. The product information sheet does not seem to mention it.
Protease inhibitor cocktail must be added fresh to the RIPA lysis buffer because protease inhibitor cocktail which is a blend of chemicals called proteases inhibitors protect proteins from degradation by endogenous proteases.
So, they are essential to most cell lysis and protein extraction procedures. If you are interested in phosphorylated proteins in the cell lysate, in addition to protease inhibitor cocktail, you need to add phosphatase inhibitors like sodium orthovanadate and sodium fluoride to inactivate endogenous phosphatases and protect protein phosphorylation.
Best.
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