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Similar peaks for dsRNA and dsRNA-protein complexes

Posted: Wed Mar 02, 2011 10:47 pm
by sheri
I'm doing a size-exclusion chromatography and am trying to separate out the dsRNA-protein complexes from a solution that has free protein, free dsRNA and complexes. Although I can see a peak for the dsRNA alone after the dsRNA-protein complex peak, I want to know why (in relation to Beer's law) the peaks are similar, i.e, both peaks have a higher 260 absorbance than 280. For protein-only peaks, the 280 is higher than 260.

Re: Similar peaks for dsRNA and dsRNA-protein complexes

Posted: Thu Mar 03, 2011 8:41 am
by HW Mueller
Most likely the peaks are not what you think they are. The Beer-Lambert law is innocent.

Re: Similar peaks for dsRNA and dsRNA-protein complexes

Posted: Thu Mar 03, 2011 3:21 pm
by sheri
The peaks are what I think they are because we done other confirmation tests which proved it. However I do not understand why the peaks are similar for the protein-nucleic acid AND nucleic acid but diff from a protein only peak.

Re: Similar peaks for dsRNA and dsRNA-protein complexes

Posted: Thu Mar 03, 2011 5:03 pm
by HW Mueller
Most likely the confirmation tests are not what you think they are.

Re: Similar peaks for dsRNA and dsRNA-protein complexes

Posted: Thu Mar 03, 2011 9:43 pm
by dorota
Proteins conformation (secondary, tertiary, and quaternary structure) has impact on absorbance. 280 nm is characteristic wavelength for aromatic rings in hydrophobic residues of proteins. Unfolding leads to an increase in absorbance. Is it possible that in your dsRNA-protein complexes hydrophobic residues are more shielded than in free protein?

Re: Similar peaks for dsRNA and dsRNA-protein complexes

Posted: Fri Mar 04, 2011 7:02 pm
by sheri
Thanks Dorota, by electron microscopy we are not able to tell, however this may be a possibility. Thanks for the input!