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Bradford's method

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Bradford's method

  1. 1. Bradford’smethod
  2. 2. 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).
  3. 3. 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
  4. 4. 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

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  • ShahzaibAli70

    Jul. 15, 2020

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