his-tagged protein that won't elute
klenchin at REMOVE_TO_REPLY.facstaff.wisc.edu
Fri Oct 6 21:23:51 EST 2000
mlsulliv at facstaff.wisc.edu ("Michael L. Sullivan") wrote:
:I'm expressing a his-tagged protein in e. coli that forms insoluble
:inclusions. I've started trying to clean up the insoluble fraction by
:using Ni-NTA agarose. So far in pilot experiments done "batch" style in
:microfuge tubes (I'm using about 50 ug protein with 25 ul matrix in a
:volume of 250 ul), I've been able to get nice binding to the Ni matrix, but
:am unable to elute it.
:I've been doing this in the presense of 8M urea during all steps. I get
:about 80% of my input material to bind at pH 8.0, followed by a pH 6.3
:wash. I've tried eluting bound protein with low pH (4.5) and with 100 mM
:EDTA (pH 8.0) with no luck. If I boil the post-elution matrix in SDS-PAGE
:sample buffer, I find the protein. I haven't tried eluting with imidazole
:Anybody have any idea what's going on? One possibility I've thought of is
:that I'm seeing non specific binding to the agarose beads, but to have it
:be so much of the protein just seems so unlikely to me.
I bet on non-specific binding. It happens. 100 mM EDTA pH 8.0 strips
Ni2+ from NTA. Thus, your protein cannot be retained specifically.
Make sure to keep high salt (~0.5 M, increasing to 1.0 won't hurt)
or, since your protein is denatured anyway, replace urea with
6-8 M GuHCl. It is stronger chaotrop, and also is an ion. Presence
of GuHCl after all Ni2+ has been stripped with EDTA
virtually guarantees you an elution. Another, perhaps more
attractive possibility, is to elute with SDS (can even use loading
buffer). Then there are various easy ways to get rid of SDS - the
choice depends on what you are going to do with the protein.
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