How many cells would be sufficient to extract cytosolic protein for western blot analysis? For instance between 1 x 10^6 - 2 x 10^6 cells?
We usally use 1*10^6 cells for WB. Depending on the cell line and how much you load on a gel it typically is enough for 3-4 Blots. However, when I had only a small amount of cells (e.g. after transfection) 200.000 cells were enough for one WB. In this case I resuspenden the cells directly in 25µl of loading buffer, boiled them and sonificated them for 1 min.
I hope I could help you
Hi Randeep,
I have been using 20 000 cells (overexpressing the protein) for one lane, the cells were lysed (boiled) directly in SDS loading buffer and loaded on a 10% gel without sonication. I would recommend optimization because the signal depends on the expression level of the protein, on the affinity of the antibody, transfer efficiency and so on...
Best wishes,
Ela
Thank you all for your responses.
I have approximately 1-1.5 x10^6 cells. But in response to Maren and Elzbieta, lysing the cells in loading buffer is a great idea that I never came across, but I will most certainly try. Just wanted to know whether you get allot of background with this approach?
Your "background" will also be determined by specific conditions confined to your experiment/choice of reagents, i.e. choice of blocking buffer, methodology (IR vs. chemi), transfer efficency etc.
If you have a moderately expressed protein and the ideal conditions surrounding western blot are met (good protein isolation, transfer etc), then your background should be minimal because your signal is relatively higher.
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