2. Aim
To determine the amount 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.
3. Reagents
0.1 N NaOH
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.
4. Sample Preparation
Weigh 100 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.
5. Procedure
Aliquots of working 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 S1 S2 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
