Human gcsf has a pI ~ 6.
As Ursula suggests you might be better off with anion exchange.
There are protocols that show you can both refold and purify onto such
column type.
Nadir
Pr. Nadir T. Mrabet
Structural & Molecular Biochemistry
Nutrigenex - INSERM U-954
Nancy University, School of Medicine
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France
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On 12/05/2010 19:17, Ursula Schulze-Gahmen wrote:
[log in to unmask]" type="cite">
Like Juergen suggested, your protein is most likely stuck to the
column, either because it never was really was refolded or because it
doesn't like to be at pH 4.5. Do you really need such a low pH to have
it bind to source 15S. Check the pI of the protein and perhaps some
different pH buffer or try an anion exchange column if possible.
Ursula
On 5/11/10 7:53 PM, megha goyal wrote:
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type="cite">
Hi all,
Our protein is gcsf. We solubilize the
inclusion bodies in 8M urea and 0.1M cysteine and then dilute it 1:20
in refodling buffer 0.1% tween 20 pH 8.2. Then we perform concentration
using proflux M12 [just concentration and not diafiltration]. Adjust
the pH of concentrate to 4.5 and load it to source 15S resin [strong
cation exchange]. The problem is we do not recover our protein on
performing IEX. HPLC and absorbance reading on concentrate show the
presence of our protein. Buffer for
loading is 25 mM na acetate pH 4.5 and elution is same buffer with 0.5
M NaCl. No protein is lost in flow thru and even 2M Nacl
washing does not show our protein. . Only when we perform NaOH wash we
do see some peak but could not analyse it as it is too alkaline and
cant run on SDS PAGE or HPLC.
What could be the reason. Where do we lose our
protein. Kindly shed some light on this on where shall I be going wrong.
thanks and regards,
meg
--
Ursula Schulze-Gahmen, PhD.
QB3, Tjian Lab
MCB, 16 Barker Hall #3204
University of California Berkeley
Berkeley, CA 94720-3204
Phone: (510) 642 8258
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