Hello, I am working with the 22Rv1 prostate cancer cell line and need to estimate the protein concentration for Western blot. I plan to lyse 1×10^6 22Rv1 cells in 100 µL of RIPA buffer (with protease and phosphatase inhibitors). Has anyone measured the protein concentration from 22Rv1 cells under similar conditions? If so, what was the typical yield or concentration?
Hello Misbah Sehar
Yes, you should expect protein concentration in the range of 300-350µg/mL under similar conditions from 1×10^6 22Rv1 cells.
Best,
Hi Misbah Sehar,
The concentration depends on the volume of RIPA buffer. I think 100 uL is a lot, for 1x10^6 cells. I would suggest that you use less RIPA, about 50-60 uL to get around 300-500 ug/ml. Bdw what conc. did you get?
Misbah Sehar ,
generally speaking, after lysing 1×10^6 adherent tumor cells (such as 22Rv1) with 100 µL of RIPA buffer containing protease and phosphatase inhibitors, the total protein extracted is about 0.5–1.5 mg, and the converted protein concentration is roughly 5–15 mg/mL.
However, the specific protein concentration will be affected by the cell growth state, lysis efficiency and buffer system. It is recommended to use BCA or Bradford protein assay to quantitatively confirm the lysate to ensure the accuracy of the subsequent Western blot loading protein amount.
The answer to this question comes from MedChemExpress (MCE) Technical Support.
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