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Biotinylation

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Biotinylation

Postby piglet1 » Thu Oct 07, 2010 7:45 pm

one question about biotinylation. After biotinyled with BIOTIN, and then lysated cells in RIPA buffer and then added strepavidin -beads for 4 hours at 4c, RUN SDS-PAGE gel, probed with the antibody I am interested, should I reprobe the blot with tubulin to proof my experient work or not??
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Postby JackBean » Mon Oct 11, 2010 8:57 am

what you mean by reprobe with tubulin?
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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Re: Biotinylation

Postby piglet1 » Tue Oct 12, 2010 4:30 pm

Normally we just detect the target proteinafter SDS-PAGE, I mean should we reprobe with anti-tubulin or actin to claim the proteins pulled down with strepavidin beads are only cell memberance fractions.

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Postby JackBean » Wed Oct 13, 2010 10:30 am

I see. yeah, you could use some controls, by which you should know they will/not be there
http://www.biolib.cz/en/main/

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Postby piglet1 » Thu Oct 14, 2010 7:24 pm

do you know any classic cytoplasmic protein marker beside Tubulin or actin.

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Postby JackBean » Thu Oct 21, 2010 6:32 am

I would guess some metabolic enzymes, like from glycolysis
http://www.biolib.cz/en/main/

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