( Theresa Redila-Flores B...)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.

5. Incubate 5 min at room temperature.

6. Measure absorbance at 595 nm.

7. For a 384-well microplate, the protein standard and diluted dye reagent volumes are reduced to one quarter of the total volume used in a 96-well plate; that is, 50 l per well.

8. For 384-well measurements, aliquot 10 l of each diluted standard into a tube.

9. Add 200 l of diluted dye reagent. Vortex for about 5 sec.

10. Let incubate for 5 min at room temperature.

11. Aliquot 52.5 l per well. Make sure no bubbles are present, as this may cause reading errors.

12. Measure absorbance at 595 nm.

Lowry Method (Bio-Rad Detergent Compatible (DC) Protein Assay) using 96- and 384-Well Plates
1. Prepare working reagent as follows: Add 20 l of reagent S to 1 ml of reagent A as needed for assay.

2. Serially dilute the standard bovine IgG protein 1:2, from 125 g/ml to 2.0 mg/ml.

3. Aliquot 30 l of each of the diluted standards into a separate 2 ml tube.

4. Add 150 l of the working reagent to each tube and then add 1.2 ml of reagent B.

5. Vortex for about 5 sec.

6. Incubate at room temperature for 15 min.

7. Aliquot 230 l of each sample in triplicate into a clear 96-well microplate.

8. Measure absorbance at 750 nm.

9. For a 384-well microplate, aliquot 6.25 l of diluted standard into a 1.5 ml microfuge tube.

10. Add 31.25 l of working reagent and 250 l of reagent B.

11. Vortex for about 5 sec.

12. Incubate at room temperature for 15 min.

13. Aliquot 57.5 l of each sample in triplicate into a clear 384-well microplate.

14. Measure at 750 nm.

Results
Bio-Rad's protein assay kit gave linear results using the Benchmark Plus microplate reader to quantitate protein
concentration. The linear range for Bio-Rads protein assay was between 50 g/ml and 1.0 mg/ml in both 96- and 384-well plates (Figures 1 and 2). Readings were linear within their designated ranges with a correlation coefficient of ≥0.99.

Bio-Rad's detergent compatible (DC) protein assay kit gave linear results using the Benchmark Plus microplate reader to quantitate protein concentration. The linear range for both plates was between 125 g/ml and 2.0 mg/ml in both 96- and 384-well plates (Figures 3 and 4). Readings were linear within their designated ranges with a correlation coefficient of ≥0.99.

Conclusion
The Benchmark Plus microplate reader is an excellent instrument to quantitate concentrations of protein samples
using 2 of Bio-Rads protein assay kits. The correlation coefficients of all the graphs were ≥0.99, within the specified detection ranges of 31.25 g/ml to 1.0 mg/ml. Assays of both
96- and 384-well plate formats gave linear graphs, thus allowing a choice of using these 2 different plate formats with excellent results along with high throughput. The results
showed that the Benchmark Plus microplate reader exhibits excellent linearity, repeatability, and accuracy for use in 96- and 384-well plate formats.

References
Bradford MM, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal Biochem 72, 248254 (1976)

Lowry OH et al., Protein measurement with the Folin phenol reagent, J Biol Chem 193, 265275 (1951)

Coomassie is a trademark of Imperial Chemical Industries PLC.

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