Lowry's method

Lowry's method

• 1.
  LOWRY’S METHOD
• 2.
  Aim To determine theamount of total protein present in the given sample (wheat flour) by Lowry’s method. Principle The amino acids Tyrosine, Tryptophan and Phenylalanine forms a complex with Cu++ in copper sulphate - sodium potassium solution to form biuret complex. This complex reduces phosphomolybdic phosphotungstic acid to hexamolybdenum blue tungstate. The intensity of the blue color can be measured at 660 nm.
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  Reagents  0.1 NNaOH  1% sodium potassium tartrate  Preparation of Lowry’s reagent  Soln. A: 2% Na2CO3 in 0.1 N NaOH  Soln. B: 0.5% CuSO4 in 1% sodium potassium tartarate  Lowry’s reagent (Reagent C): Mix 50 ml of soln. A and 1 ml of soln. B  Folin-Ciocalteau reagent (Reagent D): 1: 2 dilutions with distilled water.  Protein standard solution  Stock: 50 mg of Bovine serum albumin (BSA) dissolved in 50 ml distilled water.  Working solution: Dilute 10 ml of stock to 50 ml with distilled water.  Sample: Wheat flour.
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  Sample Preparation  Weigh100 mg of wheat flour and dissolve in 5 ml of distilled water.  Precipitate the proteins by adding 5 ml of 10% TCA. It is then kept in ice bath (refrigerator’s ice chamber can be used) for 30 min.  The tube is then centrifuged at 3000 rpm for 3 min. The pellet is washed in 3 ml of 10% TCA and centrifuged again.  The pellet is finally dissolved in 25 ml of 0.1 N NaOH.
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  Procedure  Aliquots ofworking standard (0.2-1 ml) were taken in tubes S1 to S5. 10 and 20 µl of test sample are taken in tubes T1 and T2. The volume is made up to 1ml with distilled water.  Add 5 ml of reagent C (Lowry’s reagent) to all the tubes, mix well and keep at room temperature for 10 min.  Then add 0.5 ml of Folin's reagent to all tubes, mix thoroughly and keep in dark for 30 min.  The blue color developed was read at 660 nm. A graph was drawn with concentration on X axis and OD on Y axis and is then interpolated to find the amount of protein present.
• 6.
  Reagents B S1S2 S3 S4 S5 T1 T2 Volume of working standard (ml) - 0.2 0.4 0.6 0.8 1 - - Concentration of working standard (µg) - 40 80 120 160 200 - - Volume of unknown (µl) - - - - - - 10 20 Volume of water (ml) 1 0.8 0.6 0.4 0.2 - 990 980 Reagent C (ml) <------------------------------ 5 ---------------------- ------- (Incubate at RT for 10min) Reagent D (ml) <------------------------------ 0.5 -------------------- ------- (Incubate at RT for 30 min in dark) Read at 660nm