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Protein degradation on 2D gels


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#1 barnacleman

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Posted 22 December 2012 - 12:01 AM

Hi,

Has anyone encountered protein degradation on 2D gels before?

I have sonicated my samples for 1 min (using a relatively mild blast-setting, chilled on ice, sample dissolved in urea/thiourea-based lysis buffer).

I applied the identical sonication settings for all my samples. Yet, I observed specifically that in all my 3 biological replicate control gels, protein spots were missing at the high MW, basic end (i.e. ~ 40-66KDa, pH 4-6). Nonetheless, the acidic, high MW proteins were not affected in these control gels, whilst at the same time there were no significant increase in small MW proteins (a sign of fragmented or degraded proteins).

This situation was not observed in gels from other treatments.

How would you interpret these results? Please give me some suggestions, thanks!

#2 mdfenko

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Posted 24 December 2012 - 07:58 AM

are you sure the protein is degrading and not aggregating? have you stained the first dimension to see if the protein is present and just too big to enter the second dimension or remains at the origin of the first dimension.

(and, yes, you can form aggregates in the presence of sds and/or are resistant to denaturation by sds)
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#3 barnacleman

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Posted 24 December 2012 - 09:25 PM

Hi mdfenko,

Thanks for the suggestions. Have a merry Christmas!




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