[Protein-analysis] Re: Ni binding of His tag proteins (wajahat mahmood)

Jim Young via proteins%40net.bio.net (by jyyoungjimmy from gmail.com)
Thu May 29 10:24:21 EST 2008

The wash condition is really harsh. If your proteins are sort of insoluble,
you may use urea. Otherwise you do not need urea. Regular PBS with 5 or 10
mM imidazole should be enough for your wash. You may try this bead:

By the way you may want to try this anti-His antibody:

On Sun, May 25, 2008 at 1:03 PM, <proteins-request from oat.bio.indiana.edu>

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> Date: Sat, 24 May 2008 07:38:03 +0500
> From: wajahat mahmood <wajih76 from hotmail.com>
> Subject: [Protein-analysis] Ni binding of His tag proteins
> To: <proteins from magpie.bio.indiana.edu>
> Message-ID: <BAY107-W396E5B8CDA5088265C2CEFB3C00 from phx.gbl>
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> hi everyone,I am trying to purify a His Tag protein by passing it over the
> nickel column but too much of the protein actually flows thru and does not
> bind. there is also non specific binding as i see too many bands in my
> washings. i  am using 10mM imidazole, 8M urea nad 20mM Tris as washing
> buffer and 500 mM imidazole, 8M urea  and 20 mM Tris in my elution buffer.
> Can anyone plaease tell me about the volume of Ni-NTA to be used for binding
> a specific volume of proteins in a solution. I would appreciate any ideas
> and any possible soliutions thanks and regardswaji
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