( 5. Incubate 5 min at room temp...)Benchmark Plus Microplate Reader: Quantitation of Protein Concentration Using Two Different Colorimetric Assay Kits, Rev A

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

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