Bradford protein determination; calibration curve

Dima Klenchin klenchin at facstaff.REMOVE_TO_REPLY.wisc.edu
Mon Dec 20 17:26:09 EST 1999


:
:P. Oranje wrote in message
:> At the moment I am purifing peroxidase from horseradish
:> roots. Protein determination is performed according to
:> Bradford's assay. Can someone tell me what the general
:> mathematical formula is that describes the correlation
:> between protein concentration and extinction best when
:> using Bradford's assay for protein determination ?
:
:What are you using to calibrate your protein concentrations?  Why not use
:Horseradish peroxidase, that you have bought from Sigma!  I guess that might
:defeat the object.  By extinction - do you mean absorbance?  Or extinction
:coefficient?  There is A=e c l, where A is absorbance of sample, e is the
:molar extinction coefficient, c is concentration and l is pathlength...
:Beer Lambert I think it is called...
:

The most linear results are obtained if not absorbance at 595 nm is 
measured but a ratio of A(620)/A(425). Also such calibration is less
sensitive to various interferences usual to Bradford assay.

        - Dima
 




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