I treated MDA-MB-231 cells in suspension with electroporation and drug (~3 million cells per treatment). I prepared cell lysate and performed BCA protein assay to know that I would need 200 ul of cell lysate to get 20ug of protein content to perform Western blotting. However, this will overload the SDS gel page. What should I do to avoid overloading and get good protein content?
What kind of lysis buffer do you use?
RIPA buffer should be recommended, but if you cannot harvest enough amount of protein concentration, you can increase the ratio of SDS in the lysis buffer.
Further, enough sonication is necessary.
Hello Go J Yoshida,
Thank You for your answer. I have used RIPA buffer and done sonication to prepare the cell lysate. Moreover, I didn't stimulate my cells with TNF-a or any other stimulator. I am not sure if that can be the reason of less protein. I am wondering if I can increase the protein content, increasing the number of cells.
Thank You.
In my opinion detergent interferes with BCA Reagent. Why don't u try Bradford assay and see if there is less protein to your control. You can scale up your cells if the estimated O.D is less than the lower concentration of the standard.
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