Bradford protein determination; calibration curve

Dr. Peter Gegenheimer PGegen at UKans.nolospamare.edu
Fri Dec 17 15:12:43 EST 1999


On Fri, 17 Dec 1999 16:57:13, H.V.Taylor 
<harold.taylor.delete-this at remove-this.uni-tuebingen.de> wrote:

ð >P. Oranje wrote:
ð >> At the moment I am purifing peroxidase from horseradish
ð >> roots. Protein determination is performed according to
ð >> Bradford's assay. Can someone tell me what the general
ð >> mathematical formula is that describes the correlation
ð >> between protein concentration and extinction best when
ð >> using Bradford's assay for protein determination ?
ð >
ð You'll find that fitting your results to a hyperbolic function 
ð i.e.,  Ext. = (protein * factor1) / (protein + factor2)
ð works quite well.

To answer the original post: if you are asking for a predetermined formula 
that links protein concentration to absorbance, there is none. That is, 
although you could calculate the concentration of bound dye from its 
extinction coefficient shift (bound vs unbound), you would not know how many 
dye molecules are binding to each protein molecule, so you could not calculate
the protein concentration. 

This may be too basic, but one can calculate concentration from extinction 
coefficient only if it is the compound you're measuring which is absorbing; 
it's spectrophotometrically pure, and the extinction coefficient is known. 
Otherwise, you must determine concentration by an indirect assay, or reaction 
of the test compound with a detection reagent. This reaction must be 
calibrated each time its performed, because the output (absorbance, for a 
Bradford assay) is subject to experimental fluctuations. 

To answer the follow-up: If you are using an indirect assay, you can only use 
the portion of the calibration curve which is linear. If the experimental data
(Abs vs [standard protein]) do not fit a straight line, then your assay is 
*not working*. Any assay which measures the product of a reaction between a 
reagent and the "unknown" sample *must*, by definition, exhibit a linear 
relationship between the concentration of sample and the amount of product. In
other words, you must have a linear dose-response curve, or substrate-product 
curve. 

If the relationship is not linear, you would first suspect that the reaction 
is not going to completion; that is, reagent concentration is limiting. This 
is especially true for the Bradford assay, which is (in principle) a simple 
binding isotherm. 

[On the other hand, a curve of Abs vs [dye] must have a hyperbolic shape 
because its a binding isotherm. From this you could measure Kd but you 
wouldn't want to use it for a calibrations curve of dye concentration!]

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