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SDS-PAGE resolution


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3 replies to this topic

#1 Chethan Jd

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Posted 28 November 2010 - 12:38 AM

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.

#2 bob1

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Posted 28 November 2010 - 03:01 PM

How big are the proteins you are looking at?

#3 Chethan Jd

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Posted 28 November 2010 - 10:17 PM

How big are the proteins you are looking at?

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

#4 mdfenko

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Posted 29 November 2010 - 12:35 PM

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.
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