Hi everybody. After purification of a membrane fraction, I have measured the absorbance at 215nm (peptide bond absorption) and at 280nm (absorption by aromatic amino acids (tyrosine, tryptophan and phenylalanine)). Now, at 215 nm the peak corresponding to the monomer is sharper than at 280 and the peak corresponding to the aggregates almost disappears. THe preparation os identical in both measurements. Any hint on why this is happening? And which measurement at this point is most reliable? Thanks a lot!
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DaveD
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Posted 11 February 2010 - 09:31 PM
How exactly are you going about preparing the membrane fraction? Are you talking about Bligh & Dyer (organic solvent extraction) or a cell disruption with differential centrifugation? If the latter is the case an you're dealing with membrane fragments, proteoliposomes and the like your 215 nm absorption should be dominated by Raleigh Scatter rather than absorption.
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My 2 ct.
Dave- Dilyx Biotechnologies
-DaveD-
Hi everybody. After purification of a membrane fraction, I have measured the absorbance at 215nm (peptide bond absorption) and at 280nm (absorption by aromatic amino acids (tyrosine, tryptophan and phenylalanine)). Now, at 215 nm the peak corresponding to the monomer is sharper than at 280 and the peak corresponding to the aggregates almost disappears. THe preparation os identical in both measurements. Any hint on why this is happening? And which measurement at this point is most reliable? Thanks a lot!
