Hi all,
I am trying to identify a receptor in primary immune cells for my protein of interest, but I am not seeing any potential bands in the elution buffer. For the immunoprecipitation (IP), I used 150 µL of cell lysate extracted from 2 × 10^7 cells and 2.5 µg of Flag-tagged protein. After washing three times with TBS, I eluted with 0.5 mg/mL 1× Flag peptide and elution buffer provided by Thermo Fisher.
I suspect that the amount of membrane protein may not be sufficient. However, the membrane isolation kit I have not been tested in primary cells and does not work well for receptors with multiple transmembrane domains.
Does anyone have suggestions on how to better extract membrane proteins from primary immune cells? Any other ideas would also be welcome! Thank you in advance!
Do you have a positive control? For example, is there a transmembrane protein you can express and extract from a sample that was cultured together with your primary cells?
Since I try to discover its corresponding receptor (unknown), I have no idea what kind of membrane protein will be discovered.
What was the lysis buffer composition and protocol used? M-PER reagent is useful in most cases to extract most of the membrane proteins...
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