Lab Activity 7 IUG, Fall 2017 TMZ

Determination of Protein Concentration by Bradford Method

Principle The Bradford assay involves the binding of Coomassie Brilliant Blue G-250 dye to proteins (Bradford 1976). The dye exists in three forms: cationic (red), neutral (green), and anionic (blue) (Compton and Jones 1985). Under acidic conditions, the dye is predominantly in the doubly protonated red cationic form (Amax = 465 nm). However, when the dye binds to protein, it is converted to a stable unprotonated blue form (Amax = 595 nm). It is this blue protein-dye form that is detected at 595 nm in the assay using a spectrophotometer or microplate reader.

Dye-Binding ( Bradford ) Assay CBBG primarily responds to arginine residues (eight times as much as the other listed residues) CBBG binds to these residues in the anionic form Absorbance maximum at 595 nm (blue) The free dye in solution is in the cationic form, Absorbance maximum at 465 nm (red). Bradford, MM. A rapid and sensitive for the quantitation of microgram quantitites of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72: 248-254. 1976. Stoscheck, CM. Quantitation of Protein. Methods in Enzymology 182: 50-69 (1990).

The spectrum from unbound (red line) and protein bound (green line) Coomassie® Brilliant Blue. After binding the absorbance maximum of the dye shifts from 465 nm to 595 nm.

Reagents Bradford reagent: Dissolve 100 mg Coomassie Brilliant Blue G-250 in 50 ml 95% ethanol, add 100 ml 85% (w/v) phosphoric acid. Dilute to 1 liter when the dye has completely dissolved, and filter through Whatman #1 paper just before use. The Bradford reagent should be a light brown in color. Filtration may have to be repeated to rid the reagent of blue components.

Preparation of test samples for the Bradford protein assay. Sample Volume, µl Vol. Water, µl Vol. Bradford Reagent, µl Blank 800 200 BSA Standard - 5 µg/ml 10 790 BSA Standard - 10 µg/ml 20 780 BSA Standard - 15 µg/ml 30 770 BSA Standard - 20 µg/ml 40 760 BSA Standard - 25 µg/ml 50 750 Protein Sample

Procedure 1. Prepare a 4-fold dilution of a 2 mg/ml BSA sample by adding 50 µl of 2 mg/ml BSA to 150 µl of dI water to make 200 µl of 0.5 mg/ml BSA. 2. Generate test samples for blank, BSA standards and the protein sample to be tested according to table 1 in disposable cuvettes. 3. Measure the absorbance of each sample at 595 nm using a UV-visible spectrophotometer. 4. Plot the absorbance of each BSA standard as a function of its theoretical concentration. The plot should be linear. 5. Determine the best fit of the data to a straight line. Determine the unknown protein’s concentration.