4 replies to this topic

[dnai]

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 is identical in both measurements. Any hint on why this is happening? And which measurement at this point is most reliable? Thanks a lot!

[DaveD]

dnai, on Nov 17 2009, 01:15 PM, said:

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 is identical in both measurements. Any hint on why this is happening? And which measurement at this point is most reliable? Thanks a lot!

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.
My 2 ct.
Dave
Dilyx Biotechnologies

[dnai]

Dave- THanks for your answer. Actually at 215 nm I have only one peak corresponding to the monomeric fraction and at 280 I see an additional peak for the aggregates. The peak for the monomeric fraction is comparable in the 2 measurements. That would rule out the Releigh Scatter you were mentioning. I would like to understand why the 215 looks better and why I see more aggregates at 280. I prepare the membranes with cell disruption and sequential centrifugations.

DaveD, on Nov 18 2009, 10:09 PM, said:

dnai, on Nov 17 2009, 01:15 PM, said:

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 is identical in both measurements. Any hint on why this is happening? And which measurement at this point is most reliable? Thanks a lot!

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.
My 2 ct.
Dave
Dilyx Biotechnologies

[DaveD]

dnai, on Nov 19 2009, 09:10 AM, said:

Dave- THanks for your answer. Actually at 215 nm I have only one peak corresponding to the monomeric fraction and at 280 I see an additional peak for the aggregates. The peak for the monomeric fraction is comparable in the 2 measurements. That would rule out the Releigh Scatter you were mentioning. I would like to understand why the 215 looks better and why I see more aggregates at 280. I prepare the membranes with cell disruption and sequential centrifugations.

DaveD, on Nov 18 2009, 10:09 PM, said:

dnai, on Nov 17 2009, 01:15 PM, said:

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 is identical in both measurements. Any hint on why this is happening? And which measurement at this point is most reliable? Thanks a lot!

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.
My 2 ct.
Dave
Dilyx Biotechnologies

That's puzzling. Let us know once you get to the bottom of this.
Dave
Protein solubility kits

[Prep!]

if u are talking abt HPLC measurement.. why dont u collect the peak and run an SDS gel with the standard protein and find out if its your protein or something else.. or much better is a western with polyclonal!!