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

[Jim Young]

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:
http://www.genscript.com/product_001/kit/code/L00295/category/kit/Ni_Charged_Magnetic_Beads.html?src=forum

By the way you may want to try this anti-His antibody:
http://www.genscript.com/anti_his_mab.html?src=forum
http://www.genscript.com/western_tech.html?src=forum



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

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> Message: 1
> 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>
> Content-Type: text/plain; charset="iso-8859-1"
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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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