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 (λmax = 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 λmax at 595 nm (On binding to protein the λmax is shifted from
465 to 595 nm).
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 A550 against water blank.
The A550 should be 1.1. If not adjust the dilution.
Sample: Serum
 Reagents B S1 S2 S3 S4 S5 T1 T2
Volume of working standard (µl) - 10 20 30 40 50 - -
Concentration of working - 2 4 6 8 10 - -
standard (µg)
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