Elimination of low-range co-purified proteins

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Pasteurian
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Elimination of low-range co-purified proteins

Post by Pasteurian » Tue Mar 13, 2007 2:29 pm

Hi,
I intend to purify an approximately 100-kDa recombinant protein that in my initial attempts, is co-purified with lower molecular weight proteins (20-50 kDa range) which are present in higher concentration than my protein of interest on SDS-PAGE gel. I used a 50-kDa CO centrifugal filter but it didn't help.

I would appreciate it if anyone could suggest procedure(s) or product(s) which could eliminate the lower Mw co-purified proteins.

With many thanks for your earliest attention,

P/

Jelanen
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Post by Jelanen » Wed Mar 14, 2007 11:43 pm

If you're running a SDS-PAGE column why are your smaller proteins even still around? They should be long gone by the time a 100kDa fraction comes out. Honestly, I'd start over and put an affinity tag on the protein and use affinity columns.
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Sheira
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Post by Sheira » Thu Mar 15, 2007 9:13 am

How do you purify your recombinant protein?

Vagabond
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Post by Vagabond » Thu Mar 15, 2007 4:27 pm

Your sizes may be to close for the typical membrane centrifugal filter to separate. They are not as good as they say. Call the tech support to check.

Run a size exclusion column (HPLC - preferred) or by gravity. If gravity you will want a tall column.
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