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
