Yes, it is possible that the disulfide is just re-forming when the TCEP
is gone. pH 4 is not favorable for the oxidation, but does not prohibit
it either. Especially if there are traces of metal ions around. I
learned this the hard way, as trace metals can catalyze the oxidation of
methionine and selenomethionine as well. In my hands, adding a pinch of
EDTA (~1 mM final conc.) to the fraction coming off the HPLC stabilized
the reduced species. Since the HPLC I was using at the time was made of
metal (and showed signs of rust around some of the fittings), it is
perhaps not surprising that I had a few ppm of Fe++ in the mobile phase.
-James Holton
MAD Scientist
megha goyal wrote:
Hi all,
My protein is relatively pure except the dimer. i used 10 mM TCEP to
reduce it (directly added 1 M TCEP to make final volume of 10mM and
kept at room temperature for 10 mins] then i dialysed the protein
sample to remove TCEP [dialysis buffer is Na acetate pH 4.0].
On performing SDS PAGE analysis after dialysis of the protein we are
getting no dimer band, only band of our protein is observed and same
is the case with UV reading there is no change in it. But the HPLC
analysis of the protein shows two peaks instead of one peak as
observed before TCEP treatment. what can be the reason for this.
Kindly guide. i need the protein to formulate and conduct stability
studies on the sample. the protein we obtain after IEX is pure except
the dimer and i do not want to go for SEC as it greatly reduces
protein content and also is quite time consuming. any light on what is
happening will bevery useful.
thanks and regards
