band shift in SDS PAGE with mercaptoethanol

[Dr Engelbert Buxbaum]

Am 02.08.2007, 05:24 Uhr, schrieb MarkusM <muellner.markus from gmail.com>:

>
> In a recent SDS-PAGE experiment I saw a very strange (and reproducable)
> phenomenon:
>
> BSA (and also HSA) boiled with 5% beta-mercaptoethanol (5min, 70°C)  
> showed a
> higher molecular weight (around 66kD; so this should be correct) but BSA  
> (or
> HSA) not boiled with 5% 2-ME had a band at around 55kD.

Boiling in BME (or better DTT) leads to the reduction of disulphide bonds  
in the protein, allowing the protein to completely unfold. If you leave  
out this step your protein has some tertiary structure left, which changes  
its molecular radius and hence its running behaviour. Note that it is the  
radius, not the MW, that determines the position of a protein in a  
SDS-PAGE gel.