I have been using RIPA lysis buffer (50mM Tris, 150mM NaCl, 1mM EDTA, 1%Triton-X, 1tab protease inhibitor). I am having problems detecting endogenous alpha synuclein so I want to make sure I am using the correct buffers for WB procedure.
http://tools.lifetechnologies.com/content/sfs/manuals/KHB0061_revA7.pdf
Read this.
Rao
first : i think you need to add MG132 (proteasome inhibitor) to the RIPA buffer see in (google RIPA buffer recipe) + do sonication for your lysate
second run a series of wells each well have different amount of total cell lysate (10ug to 50ug) and do incubation with your first antibody in 4c overnight in the desired dilution..
Hello Velmarini,
Did you figure out how to extract alpha synuclein from SH-SY5Y cells? Right now, I am trying to do the same, but I've learned that endogenous aSynuclein levels are too low in SH-SY5Y, and it is very difficult to detect it without transfection or differentiation.
Some follow up would be great. Thanks a lot in advance!
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