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
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