We are interested in purifying a protein (from detergent extracted microsome
preps) which binds a particular peptide.
since the pka of the peptide was in the 3-4 range, chose affigel 15 and couple
the peptide in a HEPES buffer and after washing quenched the reaction with a
I can succesfully elute about 10 bands of protein with salt (150 mM).
In order to determine the specificity I perpared a 6 mM solution of the peptide
in binding buffer (MES 100 mM 1 % CHAPS detergent) after binding the protein
and washing the column I added 2 ml of the elution buffer (6 mM peptide in
wash buffer) and the column (1 ml) swelled up to 5 ml and would not settle.
By means of a syringe I drew out the elution wash the column with wash buffer
and re-eluted with 150 mM salt. When I analyse the samples by SDS page.
The peptide elutions have a large percipitate in the presence of Laemmli
sample buffer) all the other fractions do not. Amd when the gel was stained
with silver I could not see any protein. well there are faint smears here and
there but nothing sharp and crisp.
anyone can help me?
the matrix is "clogged" all operations now have to be performed by syringe.
gravity does not work anymore.
please reply to
venkata1 at pilot.msu.edu
thanks a million
Home and lab address:
Sridhar Venkataraman phone : 24 hours : 517-353-3519 (work)
DOE-Plant Research Laboratory godly hours : 517-355-1266 (home)
122 Plant Biology Building
Michigan State University fax : 517-353-9168
East Lansing , 48824-1312 e-mail : venkata1 at pilot.msu.edu
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