Hello, everyone.~
I'm trying to do western blot for LAMP-1 protein. The cell line is 50b11 cell line(rat DRG cells). At first time, I used MPER buffer(thermofischer) and LAMP-1 monoclonal antibody from invitrogen, but didn't work.; In the manual of that antibody, MPER buffer is listed as one of the applicable lysis buffer, but I couldn't get any bands. I tried a couple of times and also IP lysis buffer(thermofischer), but all failed. I thought the antibody might be the problem for our cell line, so I tried another antibody from DSHB.(I saw a few comments recommend DSHB antibodies here) However, the results didn't change. Is there anybody who has a recommendable lysis buffer recipe for LAMP-1 western blot ??
Thank you~!
Jun.
Hi
I am not familiar with the lysis buffer that you use, but if you think that MPER buffer is not strong enough you can try RIPA buffer. Maybe you don't detect LAMP1 in WB because is very low expressed in that particular cell line. Anyway, I suggest you to validate your antibody treating your cells with sometjhing which will block lysosomes turnover (e.g. chloroquine). In this way you should see more LAMP1 protein in treated cells.
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