Instead of trying to figure out your CnBr problems, may I strongly
recommend you to use your RediPack column with Glutathione Sepharose 4B
(if you used a column to purify the protein from the total E.coli
extract) or to pack a column with your Glutathione Sepharose 4B slurry
(if you used bulk beads)? At the final step of your GST-X protein
purification from the E.coli extract, you do not elute (with reduced
glutathione) the GST-X fusion protein from the column, you keep it there
and use it for the affinity experiments, including retention and elution
of interacting proteins in a salt gradient. In addition, some of the
GST-X proteins are unstable and it helps to cut down the time of the
experiment (to say nothing of the CnBr...).
If your protein is already eluted and you do not want to start an E.coli
culture and sonication again, you can simply reload your protein on the
Glutathione Sepharose column it was eluted from (a Sephadex G-25 column
or similar method may be needed as a preliminary step, if you think that
the reduced glutathione, m.w. 300, is still present in your protein
preparation).
Best regards,
victor
www.alphadna.com
________________________
Roger Pearson wrote:
Dear ABRF members,
I have purified a recombinant GST fusion protein from
E.coli inclusion bodies (~ 50 kDa in size). The protein is presently in
solution in a sodium bicarbonate/ 8M urea buffer pH 8.3 and has been
dialysed against the same buffer, previously deionised with a mixed bead
resin. When this protein was mixed with CnBr activated Sepharose 4B no
coupling occurred. Can anyone pinpoint the problem (I am sure there were
no
free amines present in the buffers) Has anyone suggestions on how to
maintain solubility of this recombinant protein and achieve coupling to
the
Sepharose affinity resin.
Thankyou for your help.
Roger Pearson.
Mr Roger Pearson
Tropical Agriculture
CSIRO Long Pocket Laboratories
Indooroopilly Brisbane Qld
Australia.
Tel: 07 321-42790
Fax: 07 3214-2881