[Protein-analysis] Re: help with a 20 kDa protein

Dr Engelbert Buxbaum via proteins%40net.bio.net (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  
mans substitute.

> 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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