Determining Fab CONCENTRATION accurately

Mon Mar 28 14:54:19 EST 1994

I have generated Fab fragments using Papain cleavage of three different
antibodies:  one is an IgG2b and the other two are IgG1's.  Does anyone
know of an assay to accurately determine the concentration of the Fab
fragment?  Note that this is the only product in solution (I purifies the Fab
using protein A and FPLC). 

(I mean, purifieD)

I have been using an equation I got out of a crystallography text based on the
amino acid sequence and the A280 reading (you count Trp and Tyr residues and, 
based on A280, come up with a mg/ml concentration).  But I don't know how
accurate this is.

Can someone tell me of a better way, or is what I'm doing ok?  Also, is using
an IgG standard (such as from Biorad) and doing a standard curve going to
be accurate?  I mean, since the standard is a whole antibody and I'm using
Fab, is the use of IgG as a standard really comparable?

Any suggestions would be appreciated.

Rob Willard
The Wistar Institute of Anatomy and Biology
The University of Pennsylvania
Dept. of Chemistry

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