[Protein-analysis] Re: help with a 20 kDa protein
Dr Engelbert Buxbaum
(by engelbert_buxbaum from hotmail.com)
Fri Jan 30 09:26:47 EST 2009
Am 25.01.2009, 13:40 Uhr, schrieb seifollah Azadi
<sazadi from interchange.ubc.ca>:
> Last time I ran the same sample twice and side by side using DTT and
> 2ME. The lane that had been reduced by DTT showed a better result
> whereas the one with 2ME showed a kind of diffused band. I ran this
> western very short, like half an hour. I think this might be another
> reason that I got the band.
That is not untypical, DTT (Cleland's reagent) was designed specifically
to have the right redox potential for this purpose. bME is only a poor
> I know that both 2ME and DTT do not run with the protein and they are
> left behind in the top of the gel and if the protein is sensitive to
> oxidation, will be gone.
> I heard there is other reducing agent which travels all the way with the
> running protein.
Both are uncharged and hence do not move in an electrical field. Usually
proteins run fine once they have been reduced, for the few cases where
re-oxidation by air is a problem you can use TCEP
(tris-(2-carboxyethyl)phosphine) to permanently modify the SH-groups. Note
that this can not be used prior to IEF.
> 2) Why do you think when I run the gel longer the band gets disappeared?
I assume you use SDS-PAGE for separation. This is not an equilibrium
technique (unlike IEF), all proteins leave the gel eventually if you run
it long enough.
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