?Protein Quantitation?

Frances f-weis-garcia at ski.mskcc.org
Wed Sep 16 08:52:45 EST 1998


Please help -

We are currently quantitating our antibody concentration by two methods
-
(1) Comassie Blue G250/densitometery/Computer analysis (BioRAD's
Molecular Analyst Program) - and - (2) the a Bradford based assay
(BioRAD
Protein Assay) - and the two methods do not always give the same
antibody
concentraion -

When the monoclonal antibody is impure (25-45% from bioreactor
supernatant) the two approached agree very well when I correct the
bradford asssay numbers for the puritiy - determined by densitomety -
But
as we purify the antibody to 95% pure - we progressivly get less
agreement
between the two methods - to the point where the bradford assay gives
values that are at least 1/2 the value calculated from the comassie
stained
gel - We use dilutions of the same rat or bovine IgG prep (from BioRAD)
as
standards for BOTH assays -

I tend to trust the comassie staining more - because my understanding is

that comassie staining is protien composition INDEPENDENT and the
bradford system is dependent on the number of tyrosines in the protien
solution -

Therefore - does anyone know of another way to quantitate the
concentration on a purified protien that is independent of the protien
composition - or am I wrong and should trust the bradford system more -

Thanks for any advise or comments you can offer -

Frances

*************************************

Frances Weis-Garcia, Ph.D.
Manager, Monoclonal Antibody Core Facility

Memorial Sloan-Kettering Cancer Center
1275 York Avenue - Box 341
New York, New York  10021

Phone:  212 - 639 - 2054
FAX:  212 - 794 - 4019
e-mail:  f-weis-garcia at ski.mskcc.org







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