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1.  Lowery’s Protein
Quantification method …

a
M.Phool
Badshah

2.  Aim:
o To determine the amount of total protein present in the
given sample (wheat flour) by Lowry’s method.
Principle:
o The Principle of this method is based on two reactions
leading to colored complex formation. First, Lowry’s
protein assay use coppers, Copper binds with peptide
bonds in proteins under alkaline conditions, resulting in
their reduction to cuprous ion.
o 2nd, Reduction of folin’s- Ciocalteu reagent by the copper
peptide bond complex, which subsequently causes a
color change of solution into blue with absorption in the
range of 650 to 750 nm detectable with an
spectrophotometric .

4.  Reagents:
o 0.1 NaoH.
o 1% sodium potassium tartrate.
o Preparation of Lowry’s reagent.
Solution A : 2% Na2Co3 in 0.1 NaoH.
Solution B : 0.5% CuSo4 in 1% sodium potassium tartrate.
Lowry;s reagent (reagent C) : Mix 50ml of soln A and 1ml of soln B.
Folin-Ciocalteau reagent (reagent D) : Dilutions 1:2 with distilled water.
o Protein standard solution.
o Stock : 50mg of Bovine serum albumin (BSA) dissolved in 50ml distilled
water.
o Working solution : Dilute 10ml of stock to 50ml with distilled water.
o Sample : Wheat flour.

5.  Sample preparation:
o Weigh 100 mg of wheat flour and dissolve in 5ml of
distilled water.
o Precipitate the proteins by adding 5ml of 10% TCA. It
is then kept in ice Bath ( refrigerator’s ice chamber
can be used) for 30 min.
o The tube is then centrifuged at 3000 rpm for 3 min.
The pellet is washed in 3ml of 10% TCA and
centrifuged again.
o The pellet is finally dissolved in 25ml of 0.1 NaoH.

6.  Procedure:
o Aliquots of working standard ( 0.2-1 ml) were taken in
tubes of S1 to S5. 10 and 20 micro-liter of test sample
are taken in tubes T1 and T2. The volume is made up to
1ml with distilled water.
o Add 5ml of reagent C (Lowry’s reagent) to all the tubes
mix well and keep at room temperature for 10 min.
o Then add 0.5 ml of Folin’s reagent to all tubes mix
thoroughly and keep in dark for 30 min.
o The blue colour developed was read at 660 nm . A graph
drawn with concentration of X-axis and OD Y-axis and is
then interpolated to find the amount of protein present.

8. Advantages:
 It is sensitive assay which require no digestion of
protein.
 It is more specific.
 It is simple to perform and can be easily used on
small scale in labs.
 It is significally more sensitive.
 It is 10 or 20 times more sensitive as compared
with ultraviolet absorption at 280 nm.

9. Limitations:
 Sensitive to PH changes and therefore the PH of
assay solution should be maintained at 10-10.5.
 Sensitive to low concentration of protein.
Concentration of protein should range from 0.10
to 2mg of protein per ml.