Hello,
I would like to run a western blot for ATR signaling protein.
I am using U2OS cells.
Anybody knows the minimum amount of cells that I can use for that?
Are 1.5 million of cells enough to have a good signal?
Thanks in advance
hi
It depends on the number of tests and your needs (usually this number of cells is enough to load in 10 wells). It is best to determine the concentration of protein in cell lysates. This will give a more accurate and clean result (usually 20 micro grams per well).
"1.5 million of cells in 150ul RIPA lysis buffer"
https://www.abcam.com/protocols/sample-preparation-for-western-blot
https://www.thermofisher.com/order/catalog/product/23225#/23225
*** In my opinion, 5 million cells is better if you need more tests and repeat them.
good luck
Hi,
The order of magnitude is generally 1-5 mg/ml protein in 200 µl lysis buffer on a 10 cm dish that typically contains 5 x 10e6 cells. The limitation to scale down protein extraction is the concentration of the extracts vs the volume of the SDS-PAGE wells to load.
In small SDS-PAGE gels you can go down to 10-15 µg if your protein is abundant enough and if your antibody is good but 20-50 µg is better. And if 10 wells are enough for the nb of conditions you have you can load 50 µl in 1.5 mm thick gels.
Good luck
Daniel
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