I need at least 100ug of total protein of LNCaP cells. Can I use T25 flask with 60-70% confluency stage or do I need to wait until it becomes more confluent? How much RIPA is preferable for lysis?
A lot of variables are going to factor into this such as efficiency of lysis, efficiency of harvest (either by trypsin or scraping), etc.
Personally, I do not believe even under optimal conditions will you achieve 100ug total protein. I would estimate if you were to harvest the cells, lyse in ~20-30uL RIPA, you would get 1-2ug/uL, so at best 60ug total (this is just a pure estimation on a lot of experience harvesting protein).
I would suggest either splitting the flask, as opposed to allowing it to become more confluent. Confluence changes properties of cancer cells, and will convolute your interpretation.
As far as the lysis goes, it depends on your assay. If you are doing a simple western blot then RIPA will be fine. If you are doing a protein activity assay, use something less denaturing or harsh. You can use sonication as well to lyse the cells if you have access to a sonicator, but beware that this also may affect your downstream assay depending on what it is.
Good luck,
~Adam
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