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Protein conjugation affecting motility (Double bands)

Protein conjugation affecting motility (Double bands) - SDS-PAGE Gel Electrophoresis Forum

Protein conjugation affecting motility (Double bands) - SDS-PAGE Forum and Gel Electrophoresis Forum. Discuss the running of agarose gels, sds-page gels, and other gels including sequencing, gradient or tricine.


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Old 03-02-2012, 02:27 PM
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Default Protein conjugation affecting motility (Double bands)



Hi,
I'm currently treating cells with increasing concentrations of a compound, before running the lysate on an SDS-PAGE gel and probing for a protein during a Western blot.

I've noticed that i'm getting a double band appearing, which is unexpected! One of them is at about the weight I expect - 31 kDa (thought it does appear slightly lighter than the pure protein positive) but the second band is at a higher weight, around 39 kDa. Upon densitometry analysis it appears that as the concentration increases the top band gets darker and the bottom gets lighter, leading me to conclude that the compound is causing an increasing number of the protein to become modified.

My best guess is that this is a conjugate, probably GSH.

I was wondering, could a GSH conjugate decrease protein motility, making the protein appear at a heavier weight? I've been searching for papers on this for days and I can't find anything! The post-doc i'm working with keep slyly hinting that i'm on the right track, but i'm at a dead end!

I'm pretty sure it isn't protein degradation because there are only two bands and the GAPDH i'm using as a standard doesn't have any additional bands. Although, if there was increased glutathiolation wouldn't the GAPDH have double bands too?

I'm running a reduced gel, i'm pretty sure the protein is properly denatured because I went overboard on the B-ME and heating just to double check and got the same result.

Any views would be greatly appreciated

Cheers,
Forsh
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