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SDS-PAGE resolution - Problem in resolving high molecular weight bands on SDS-PAGE (Nov/28/2010 )

Hi,
I cannot resolve the high molecular weight bands on a 10 or 8% SDS-PAGE. the bands look smudgy, the samples are native and whole cell extracts of S. cerevisiae. But the marker bands resolve it properly, but not very crispy bands. I've tried preparing the reagents, buffers freshly, but things are not working.

Chethan.

-Chethan Jd-

How big are the proteins you are looking at?

-bob1-

bob1 on Sun Nov 28 23:01:39 2010 said:


How big are the proteins you are looking at?

The proteins ranging from ~40kDa to 120kDa, kindly suggest.

-Chethan Jd-

possible causes are:

aggregation of the protein

post-translational modifications (eg glycosylation)

inefficient stacking (at least 5mm in minigels and 10mm in standard or larger gels)

overloading of the protein

you can also try running a gradient gel to sharpen the bands.

-mdfenko-