Question about making low percent SDS-PAGE gels for big protein - (May/06/2006 )
I usually do SDS-Page gels with 8-12% separating gel and 5% stacking gel.
next week I need to make a 4% gel because I need to separate very well high molecular weight proteins.
My question is:
in such a low % Bis/Acr. gel, will I have still separating and stacking gel or only one gel with 4%?
Only the separating gel need to be 4%.
and which % should have the stacking gel?
your stack can be the same % or slightly lower (eg-3.5%) as long as the buffers in the polymerization mixture are at a different pH (just like with the laemmli method).
That's right, the stacking and separating gels have different pH 6.8 and 8.8, respectively. and actually I don't remember why is that
the proteins move more slowly at the lower pH, the buffer front from behind the proteins (pH 8.3 or thereabout) catches up to the proteins and speeds them up so that they catch up to the slower moving proteins and sharpens the bands for their entry into the running gel. this the stacking effect.
Where can I read more about this stacking gel effect?
I didn't understand completely how it works.
i don't know specific titles but there are probably plenty of books on gel theory available.
the lower pH of the stacking gel and sample buffer make the protein move slowly as it enters the resolving gel. the lower concentration of the stacking gel make it less restrictive so all of the proteins will move together. the higher pH of the reservoir buffer makes the proteins at the back of the load speed up as the buffer front passes through. the proteins at the rear of the load catch up to the front of the load thereby compressing and concentrating the protein load and allowing a sharp protein band to enter the resolving gel. this band will then separate by size (with some band spreading, which is why you don't have super sharp bands in your gel).
i hope that this explanation is clear. i know what i want to say, and i can see it clearly in my head, but i'm not sure that i'm saying it clearly.
Yes thanks, is more clear now.
hithat's why a stacking gel should not excess 05.cm in minigels, and 1cm in big page gels