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The assay here is designed for use in microtiter plates. This is an easy assay format for those with access to multiple channel pipettors and microtiter plate spectrophotometers.
Bradford Reagent - Bio-Rad sells a ready-to-use reagent (cat#500-0006) that can be stored at 4 C. Alternatively, the reagent can be made by dissolving 100 mg Coomassie Blue G-250 (available from several sources) in 50 ml 95% ethanol, adding 100 ml 85% (w/v) phosphoric acid to this solution and diluting the mixture to 1 liter with water.
Bovine serum albumin (BSA) (1 mg/ml) - we dissolve BSA in saline and store it frozen in 1 ml aliquots for quick use. The standard should be dissolved in a buffer similar to that the unknowns will be dissolved in. Note: other standards are acceptable and may be preferred for certain applications (for example, use an IgG standard when measuring concentrations of immunoglobulins.
Microtiter plates - we use Micro Test III Flexible assay plate from Falcon because they are cheap and work well.
Any multi-well microtiter plate reader equipped with a 595 nm filter.
Note: do all determinations in duplicate or triplicate
1. Pipette 0, 2, 4, 6, 10, 15 and 20 µl of BSA (1 mg/ml) into assigned wells of a 96-well plate.
2. Pipette up to 20 µl of unknown samples into individual wells of a 96-well plate.
3. Add 40 ml of Bradford Reagent into all wells containing standard or sample.
4. Add dd H 2O to all wells to bring the final volume to 200 µl.
5. Read absorbance at 595 nm without any prior incubation.