Bradford's method

Protocol

1. Bradford’s method

2. Estimation of protein by Bradford’s method Aim To determine the amount of protein present in the given sample (serum) by Bradford’s method. Principles The dye Coomassie brilliant blue G-250 (λ max = 465 nm) appears as a pale-orange red in protonated form (i.e.), in acid solution. It binds strongly to positively charged groups of protein and also to hydrophobic regions in protein. As a result, a blue color is formed with a λ max at 595 nm (On binding to protein the λ max is shifted from 465 to 595 nm).

3. Reagents Protein standard solution: Stock: 50 mg of Bovine serum albumin (BSA) dissolved in 50 ml distilled water. Working solution: 10 ml of stock diluted to 50 ml with distilled water. Bradford’s reagent: Stock (2X): Dissolved 10 mg of G-250 in 10 ml of absolute ethanol and placed in the shaker for 60 min. Added 10 ml of 88% O-phosphoric acid, mixed well and made up the volume to 50 ml with distilled water. Filtered through Whatman-1 filter paper. Working concentration: Diluted 1:1 with water and checked A 550 against water blank. The A 550 should be 1.1. If not adjust the dilution. Sample: Serum

4. Procedure Pipette out increasing concentrations (10µl - 50 µl) of BSA in clean dry test tubes (2 µg to 10 µg range). Make up to the volume to 100 µl with distilled water. Keep a blank. Add 1.3ml of the dye solution to each tube and mix gently. Incubate the tubes for 5 minutes at room temperature. Read the absorbency at 595 nm.

5.   Reagents   B   S1   S2   S3   S4   S5   T1   T2 Volume of working standard (µl) - 10 20 30 40 50 - - Concentration of working standard (µg) - 2 4 6 8 10 - - Volume of unknown (µl) - - - - - - 10 10 Volume of water (µl) 100 90 80 70 60 50 90 90 Dye solution (ml)   <------------------------------ 1.3 ---------------------------> (Incubate at RT for 5 min ) Read at 595nm