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Low weight protein degradation during western blot

western blot protein degradation protein detection actin Class B Scavenger Rec

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#1 kaaba

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Posted 16 May 2017 - 01:10 AM

I have problems in my last western blots performed with samples of mice liver: I observed only a strong signal underlining the band of SR-BI protein (my target), but the control protein is not detected (weak β actin signal and/or spot not defined as well as not uniform in all the samples). First I used the anti-β actin antibody, and in other experiments I incubated the liver samples with other antibodies for other proteins (such as GAPDH) in order to find a reliable control protein to compare the SRBI signals; despite these changes, I obtained the same results.

My question is: knowing that the samples have been defrosted and re-used numerous times, is it possible that the samples are starting to be degraded, in particular the low-weight proteins (β actin, GAPDH) instead higher weigh proteins, such as SRBI (75kDA ca.)?

 

Help me pleaseee ! 

thanks 



#2 mdfenko

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Posted 16 May 2017 - 03:33 AM

it's possible that you are experiencing proteolysis in your samples (liver is full of proteolytic enzymes). some proteins are more susceptible to proteolysis than others which may account for some of the differences you're seeing in degradation.

some aggregation, not reversed by sds-2me, may be occurring, as well.

 

to avoid these problems you should include protease inhibitors in your extraction medium and replenish those with short half-lives as necessary. and you should aliquot the extract into single use samples and not refreeze after use.


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