I tried buffer: 1%NP40, 20mM Tris, 137mM Nacl, 10%glycerol, 2mM EDTA, 1mM activated Na-Orthovanadate, Aprotinin 10ug/ml,leupeptin 10ug/ml. Incubation with lysis buffer for 30min but still I see the cell not completely lysing. (Adherent cells).
What modifications shall be done?
You could scrap the cell to detach them. After, you could add a step of sonication or a cycle of quick freeze/thaw.
If you would like to lyse your cells in the flask, you need a chaotropic agent like urea and thiourea but it was a different procedure.
Dear Stephane,
Thanks for reply. Since I am doing this expt. in 96well plate, scrapper wont work. Yes Freeze thaw can be a better option but how about the integrity of protein as I have to process those samples further for phospho ELISA.
Ive used both HEPES and Tris based lysis buffers similar to what you listed above and also NP40 and triton, neither of them completely lyse colonic cells without sonication. Ive had better success with RIPA (likely due to the SDS) however this may or may not work based on your application.
Dear Nikhil Shah,
I would like to buy a non-cancerous epithelial cell line. I am interested in CCD 841 and FHC. However, I am not sure what cell line to buy. If you have worked with FHC, can you please let me know your experience with this cell line?
Thanks in advance,
Juan Antonio
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