3 questions

Dr Engelbert Buxbaum via methods%40net.bio.net (by engelbert_buxbaum from hotmail.com)
Mon May 26 07:47:00 EST 2008


Am 23.05.2008, 06:17 Uhr, schrieb Kamalian, Laleh  
<L.Kamalian from liverpool.ac.uk>:


> 1. Could 2 different bands (size wise) be detected for the same protein  
> using poly colonal and monoclonal antibodies?

Sure. Any post-translational modification that changes size or charge of  
the protein results in two bands. Unless your (monoclonal) antibody is  
specific for either of these forms, you see both.

> 2.  If the samples are run on electrophoresis for too long, can the size  
> >of the protein show difference comparing to the previous (shorter) runs?

If you run the gel too long, you no longer have the front marker dye. Then  
determination of Rf values becomes impossible and MW determination  
imprecise at best.

> 3.  In siRNA , I have knock down my gene of interest at the mRNA level  
> (70-80% according to quantitative PCR x2 experiments), but western blot  
> of the same colony cell protein lysates show no or less ( only 30% )  
> decrement.  Any explanation please?

Correlation between [mRNA] and [Protein] is marginal, explaining only  
about 1/3 of the variability in [Protein]. There is a lot of regulation  
going on at the level of translation and protein degradation. See for  
example

@article{Gyg-99a,
         AUTHOR= {S.P. Gygi and Y. Rochon and B.R. Franza and R. Aebersold},
         TITLE= {Correlation between protein and {mRNA} abundance in yeast},
         JOURNAL= {Mol. Cell. Biol.},
         VOLUME= {19},
         YEAR= {1999},
         PAGES= {1720-1730},
         LANGUAGE= {engl}
}



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