SDS-PAGE - Dimer formation after boiling?

Bean Long ben.long at yourfinger.anu.edu.au
Tue Oct 3 23:12:54 EST 2006


DK wrote:
> It is a recurring subject in this group over past ~ 10 years. You can 
> probably find more details searching Google groups. In brief:
> no, it's not common but yes, some proteins do behave this way. 
> 
> Careful handling of the gel and sample usually helps to reduce
> or completely eliminate oligomerization of unfolded structures.
> I can't vote for the relative importance of the factors but this 
> combination worked for me quite nicely in the past: 
> 
> - Do not boil, do not heat at all. Use 200 mM DTT (not bME) 
> in 2X loading buffer (store frozen!) and simply incubate > 15 min 
> at room temperature.
> - Do not use glycerol in loading buffer. Substitute with sucrose.
> - Don't let the gel heat. Run it in the cold room with ice-cold buffers
> and at reduced voltage (150V constant for standard mini)
> 
> DK
> 
> P.S. SDS gels without reducing agent invariably look horrible
> because of extensive cross-linking. 
> 

Many thanks DK.  I'll give it a go.  I've also just heard from a 
colleague that using a reducing agent during protein extraction goes a 
long way to minimising polymerisation of proteins on subsequent reducing 
gels.  I would have thought that it doesn't matter until you run the 
gel.  Any thoughts on this?

Cheers and thanks,

-- 
Bean

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