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protein purification problem

Chris McDermott-Roe via methods%40net.bio.net (by cjmcdermottroe from googlemail.com)
Fri Sep 4 10:22:06 EST 2009


Hi all,

I'm having problems purifying a myc-tagged protein.  My protein of interest
is toxic in bacteria so I decided to express it in mammalian cells (HEK293s)
and (at least in theory) purify it using an anti-myc antibody agarose resin
that I got from Sigma (A 7470) and some SigmaPrep spin columns (MC1000) also
from Sigma.  Well to cut a long story short, I havent been able to isolate
my protein.  I dont know if my protein simply isnt sticking to the beads
(maybe because the tag is buried in the protein for some reason) or that I
can elute it.  If anybody has any strategy/reagent suggestions, that would
be great.

Some details that may be relevant: I've got mounds of protein as I harvested
cells and prepared proteins from 5x75 cubic cm flasks so I'm pretty sure
it's not an issue of quantity.  Second, I my protein does have the tag; I've
verified this by probing blots with an anti myc ab.  The buffer that I used
to elute is 0.1 M glycine (pH3) + myc peptide (20 micrograms/ml)

again, many thanks in advance


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