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:
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
uschulze-gah...@lbl.gov
