Nickel affinity purification (mnr mnr)

mnr mnr via methods%40net.bio.net (by mnr475 from gmail.com)
Sun Jun 5 04:23:41 EST 2011


Thanks Mark and for another off-board reply. My protein need only 20-25 mM
NaCl but I include 500 mM NaCl for nickel affinity to avoid electrostatic
interactions. 10 mM imidazole to avoid contaminant proteins.

I have tried to put N-terminus tag instead of C-terminus but it does not get
exported into the priplasm.

I will try omittig imidazole. Should I decrease NaCl to 20 mM also?

On Fri, Jun 3, 2011 at 7:26 PM, Mark Nance <mrnance from umich.edu> wrote:

> Hi Matt,
>
> With that much NaCl, you can probably omit the imidizole during the loading
> and wash steps.  One of my proteins requires 500 mM NaCl, and even though it
> has a 10-histidine tag it elutes at (or before) 50 mM imidizole.  If you
> still have your non-bound protein, you can dialyze/dilute out the imidizole
> and run it back over the column.
>
> - Mark
>
>
>
>  Message: 1
>> Date: Fri, 3 Jun 2011 11:57:40 +0100
>> From: mnr mnr <mnr475 from gmail.com>
>> Subject: Nickel affinity purification
>> To: methods from magpie.bio.indiana.edu
>> Message-ID: <BANLkTik9-6G30REknHBcWK=cgp-8LoMLkQ from mail.gmail.com>
>> Content-Type: text/plain; charset=ISO-8859-1
>>
>> Hi.
>>
>> My protein contains 6 histidine residues for His tag purification. Less
>> than
>> a mg of protein is binding whereas therest about 10 mg don't. Both bound
>> and
>> unbounds have his tag on western blot.
>>
>> I am using 10 ml of nickel sepharose from GE in 50 mM Tris pH 8, 500 mM
>> NaCl
>> and 10 mM imidazole in sample and equilibrate buffer. Protein elutes at
>> 200
>> mM imidazole. Is there any way to increase the affinity of protein
>> binding?
>>
>> Thank you.
>>
>> Matt
>>
>
>


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