Properties of proteins purified in imidazole
rozsa at UMICH.EDU
Tue Aug 10 20:06:04 EST 1999
Hi protein experts,
I'm using Invitrogen's pTrcHis vector (without IPTG and overnight growth) to
express a protein with an engineered His tag. I tried with and without IPTG
and found better expression without it, so much for induction, though maybe
this thing is toxic at too high expression levels. After trying to remove
the His tag with dubious success, gave up and am just trying to elute the
protein from the column using various imidazole concentrations. I need to
optimize the protocol on a small scale (from 100 ml of E. coli) before
moving up to larger scale. I'm using Western Breeze immunoblot
system with rabbit polyclonals. Got alot of E.coli junk so increased the
volume of pre-elution wash many fold and eluted with multiple volumes of
increasing imidazole in 25mM increases in effort to elute off E.coli
proteins and try to hit the "sweet spot" for this protein. Now my samples
there but it's in imidazole and way too dilute.
I'm a newbie in protein work so forgive the following naive questions:
1. Does imidazole interfere with SDS-PAGE? If not, can I use the blot for
subsequent western blots?
2. Anyone have any idea if imidazole is toxic to Cos7 cells. I want to see
if this protein binds to tissue culture cells. Just looking for a heads
up answer here, I know this is easy to check.
3. I can dialyze the protein out of imidazole (vs 10 mM Tris, 0.5% Triton),
but when I concentrate using an amicon filter, the sample precipitates.
What is a good buffer to store protein samples in at -80?
4. What about an ice cold acetone precipitation? Is this considered
"denaturing"? I've heard that detergents form a slimy, difficult to
solubilize pellet in acetone. Any hints about maintaining biological
integrity using acetone precipitation?
Thanks in advance!
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