My fusion protein is MBP-Dlx5 (a homeobox gene) which was purified using
amylose resin. My test proteins are Dlx3, Msx-1 and Dlaxin-1.
Thanks for the suggestions. I am going to try them. I was reluctant to try
non-ionic detergents as manufacturer suggested they might drastically reduce
binding efficiency. That might actually be the solution to my problem, if
that did not affect the specific binding of MBP fusion protein to the
amylose resin !
I need some magic that eliminates non-specific (from my perspective) binding
but does not affect the specific one.
Deepak
> ----------
> From: Artem Evdokimov
> Sent: Thursday, January 17, 2002 10:57 PM
> To: proteins at hgmp.mrc.ac.uk> Subject: Re: Tricks to eliminate non-specific binding of proteins to
> amylose resin, any ?
>> Hi,
>> You do not specify what protein(s) you work with. There is a multitude of
> reasons as to why the protein fusions may 'stick' - you'd be well advised
> to
> provide more details before we could propose anythig but pure speculation
> :)
>> Normally, MBP fusions are run in 25 mM phosphate pH 8.0, 300 mM NaCl which
> is the recommended buffer as far as manufacturer is concerned. Adding
> ethylene glycol or glycerol often helps and so does the addition of
> nonionic
> detergents.
>> A.G.E.
>> ""Khadka, Deepak (NICHD)"" <khadkad at mail.nih.gov> wrote in message
> news:000C316550E62144830CDFCEDA73050701E6282E at nihexchange4.nih.gov...> >
> > I am performing binding assay of some test proteins with MBP-fusion
> protein.
> > When I try to purify the mixture by using amylose resin, most of the
> test
> > proteins gets stuck to the resin. My buffer (both for binding and
> washing)
> > is 50 mM PIPES (pH 7.0), 100 mM NaCl and 1 mM EDTA. Playing with the
> salt
> > concentration and adding BSA did not help. Any suggestions ?
> >
> > Deepak
> > NICHD/NIH
> >
> > ---
>>>
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