help with a 20 kDa protein

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help with a 20 kDa protein

seifollah Azadi
 Hi all,
I am working with a 20 kDa protein which is kind of novel and not that much work has been done on this protein. By western blot, I some times get the band and most of the times can’t.
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.
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.
So I would be extremely appreciated if you could help:
1) Do you know any other reducing agent which can be run with the sample along the gel?
2) Why do you think when I run the gel longer the band gets disappeared?
Many thanks in advance
Seifollah Azadi


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Re: help with a 20 kDa protein

Dr Engelbert Buxbaum
Am 25.01.2009, 13:40 Uhr, schrieb seifollah Azadi  
<[hidden email]>:

> 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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