Hi everyone,
I have some cell cultures of brain tumors, which contain a high amount of dead cells after detaching them with accutase (only 30% living). I tried to isolate proteins and performed a western blot, but I didn´t get usable results. In the total protein stain I couldn´t see proteins in the line. I also tried to lyse the cells without detaching them with accutase in the bottle using lsye-buffer (RIPA with PhosSTOP and supplements) but it also didn´t work. Same problem with the western blot. Has someone an idea how I can get proteins from those cultures, which I can later use for a western blot?
Thanks a lot.
I recommend that you use another lysis buffer, in our Lab we use triton buffer plus phosphatase and protease inhibitors,
in WB it's important the size of the proteins and then make the accurate concentration of your gel.
I would first check if the amount of cells you are trying to use as starting material is enough for protein extraction. I would try to increase that amount of cell because it looks strange that after protein extraction you don't get to see any band in the lanes.
Also, have you quantified the protein concenctration after extraction?
Thank you both.
I use one million living cells for the extraction. After the extraction, I quantify the protein concentration. There is no difference between the cell cultures, which work and those, that doesn´t work. The concentration is similar. In the end I put the same amount of protein in each line.
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