SDS-PAGE - Dimer formation after boiling?
ben.long at yourfinger.anu.edu.au
Tue Oct 3 23:12:54 EST 2006
> 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)
> 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,
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