? Protein Quantitation ?
Frances
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...@ski.mskcc.org
Peter
<f-weis...@ski.mskcc.org> wrote:
> 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 -
Have you given the BCA reagent method a try? It is cheap and very easy.
Peter
--
"Don't you eat that yellow snow
watch out where the Huskies go" FZ
---------------------------------------------------------------------
Ian McFarlane
Robert Burrows
>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 -
>
Frances,
If a knpwledge of the absolute concentration of your antobody is
important, you are better off using a quantitation method that is truly
composition-independent such as a determination of total nitrogen or an
amino terminal ana;ysis. Of course, these methods are only useful with
purified proteins.
--
Robert Burrows
r...@nebiometrics.com
Pete
> that comassie staining is protien composition INDEPENDENT and the
> bradford system is dependent on the number of tyrosines in the protien
> solution -
Err.. I was under the impression that the bradford system uses
coomassie...if I remember correctly, the ORIGINAL article describing the
bradford method has a list of compounds that interfere with the assay
(Bradford, 1976 or something...).
|) |/
|etri |\ursula
-------------------------------------------------
"I am somehow less interested in the weight and
convolutions of Einstein's brain than in the near
certainty that people of equal talent have lived
and died in cotton fields and sweatshops."
-- Stephen Jay Gould
-------------------------------------------------
Cornelius Krasel
[protein quantitation: densitometric evaluation of coomassie-stained gels
vs. bradford]
> 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 -
The Bradford system *uses* Coomassie. There are proteins which stain
very badly in a Coomassie-stained gel. The Bradford system gives also
quite different readouts with different proteins, for example IgG (?)
gives about half of the absorption of the same amount of BSA.
You might want to look into Biuret or Lowry assays which, as far as I
know, are less dependent on protein composition than Coomassie binding
to protein because the colour is produced by copper complexes with the
protein backbone. There is also the BCA method, amidoblack quantitation
or -- if the amino acid composition of the protein is known and the protein
is pure -- quantitation by UV absorption.
--Cornelius.
--
/* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */
/* D-97078 Wuerzburg, Germany email: pha...@rzbox.uni-wuerzburg.de SP4 */
/* "Science is the game we play with God to find out what His rules are." */
noone
> >
> > that comassie staining is protien composition INDEPENDENT and the
> > bradford system is dependent on the number of tyrosines in the protien
> > solution -
>
> coomassie...if I remember correctly, the ORIGINAL article describing the
> bradford method has a list of compounds that interfere with the assay
> (Bradford, 1976 or something...).
>
Yep, as far as I know the only difference between Coomassie stain and
Bradford are a couple of methyl groups on the molecule, which cause a
difference in solubility. That's all...
Peter
Marco Kresin
To measure the Concentration of Monoclonals Antibody axactly for IgG there
is in the literature (Heidelberger Taschenbücher ; M. Holtzhauer
Biochemische Labormethoden Springer Verlag Page 8) a formel to
quantitating the antibody concentration:
IgG mg/ml= 1.38 *A280 in a 1cm light way
--
Marco Kresin
Fraunhofer Institut
Dep. Genetechnology
Nikolai-Fuchs-Str. 1
30625 Hannover
Germany