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Benchmark Plus Microplate Reader: Quantitation of Protein Concentration Using Two Different Colorimetric Assay Kits, Rev A

Theresa Redila-Flores, Bio-Rad Laboratories, 2000 Alfred Nobel Drive, Hercules, CA 94547 USA


The Bradford and Lowry assays are 2 colorimetric assays that are commonly used to quantitate protein concentration. Bio-Rads protein assay (catalog #500-0001), based on the Bradford dye-binding assay (Bradford 1976), measures total protein concentration. The standard assay is used for protein concentrations ranging from 50 g/ml to 1.0 mg/ml. This assay is based on the change of color of Coomassie Brilliant Blue G-250 dye with different protein concentrations, in which the dye binds to basic and aromatic amino acid residues. This assay is measured between 465 and 595 nm. Bio-Rads detergent compatible (DC) protein assay (catalog #500-0111) is similar to the Lowry assay (Lowry et al. 1951), but only requires a 15 min incubation. The reaction between protein and copper in the alkaline solution and the Folin reagent reduced by the copper-treated protein leads to color development. This protein assay is measured between 650 and 750 nm, with protein concentrations ranging from 200 g/ml to 1.0 mg/ml.


Procedure
Bradford Method (Bio-Rad Protein Assay) Using Clear 96- and 384-Well Plates
1. Dilute dye reagent with 1 part of dye reagent concentrate and 4 parts of distilled water. Filter through a Whatman #1 filter. The diluted solution is good for about 2 weeks when stored at room temperature.

2. Serially dilute standard bovine IgG protein 1:2, ranging from 31 g/ml to 1.0 mg/ml.

3. Aliquot 10 l of each diluted standard in triplicate into a clear 96-well microplate.

4. Add 200 l of diluted dye reagent. Mix thoroughly using a microplate mixer.
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