Bradford assay - (Jun/13/2007 )

Hi, im trying to generate a standard BSA curve. Here are the details;

- 0.1-1.0 mg/ml as the standard protein concentrations
- Bradford reagent is from Sigma
- 5 ul of standard proteins + 250 ul of reagent (micro assay) in microtitre plate

It seems to be quite easy to get the standard curve but somehow i didnt manage to do that. Anyone can help me with this? any better protocol?
thanks.

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What do you mean when say "i didnt manage to do that". Problem with bad calibration curve? What wavelength do you use? 595nm ?

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my protocol is
standard protein conc-- 1 mg/ml

i made dilution series from .78 ug to 200 ug.

from each diluted tube i transfer 160 ul sample soln in 96 well plate add 40 ul Commassie G dye from bio rad , shake gently wait 5 min in rt and then measure OD 595

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I use 1-10ug (total) - so 1-10ul of 1mg/ml - I use a 96 well plate. Standard is bought from sigma while the reagent is from Bio-Rad. Another thing I do that most people don't is that I also read the sample at 450nm and then divide 595/450. This increases the linearity, especially at very high and very low protein concentrations. You can have a look at the paper that published this: Zor and Selinger, Linearization of the Bradford protein assay increases its sensitivity: theoretical and experimental studies.
Anal Biochem. 1996 May 1;236(2):302-8.
Good luck

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It is intersting, but you can avoid high concentration by series dilution. And concerning low concentrations I will advise you to use BCA assay. This assay is very sensitive and give more reliable results. In my work I use Bradford only to identify protein in eluate fractions. To more accurate protein determination for protein with unknown extinction coeff, I use BCA assay

Good Luck!

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i didnt even manage to get a straight curve. yes, the wavelenght is 595nm.

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I couldnt get the paper. anyway, ill try to use the concentrations you suggested. thanks.

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Thanks.

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