Hello,
I intend to apply Mem-PER Plus Extraction Kit (ThermoFisher) for membrane protein extraction from LNCaP cell culture (adherent mammalian cells).
I tried to use this kit exactly as described by manufacturers. Then, I quantified both cytosolic and membrane fractions (Bradford Assay). However, the result was similar to those obtained for blanks.
Thus, i am looking for alternatives to solve my problem:
- Increase cell number?
- Decrease pippeting volumes of buffer solutions?
Thank you for the attention.
The detergents in the kit may be interfering with the Bradford assay. Try using the BCA assay instead.
Another possibility is that you didn't use enough cells, so there just isn't much protein there.
Thank you Adam for your answer.
I use 5 000 000 approximately, as described in users manual.
I'll try to use BCA assay, as you suggested and also increase the amount of cells.
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