UNIKL

MICET 1
CFB 20703 FOOD CHEMISTRY

EXPERIMENT 4: DETERMINATION OF MILK PROTEIN USING LOWRYS’ PROTEIN

ASSAY

The Lowry procedure is one of the most venerable and widely-used protein assays, being first
described in 1951 [Lowry et al., J. Biol. Chem. 193: 265-275 (1951)]. Under alkaline conditions,
copper complexes with protein. When folin phenol reagent (phospho-molybdic-
phosphotungstic reagent) is added, the Folin-phenol reagent binds to the protein. Bound reagent
is slowly reduced and changes color from yellow to blue.

Milk contains 3.3% total protein. Milk proteins contain all 9 essential amino acids
required by humans. Milk proteins are synthesized in the mammary gland, but 60% of the amino
acids used to build the proteins are obtained from the cow's diet. Total milk protein content and
amino acid composition varies with cow breed and individual animal genetics.

Objective

To estimate the amount of protein in milk by Lowry’s assay

Materials Required

Reagents Required:
• A: 2% Na2CO3 in 0.1 N NaOH
• B: 1% K Na Tartrate in H2O
• C: 0.5% CuSO4. 5H2O in H20
• Reagent I: 48 mL of A, 1 mL of B, 1 mL C
• Reagent II- 1 part Folin-Phenol [2 N]: 1 part water
• Bovine Serum Albumin (BSA) Protein standard- 1 mg/mL
• Fresh milk
Apparatus and Instruments
• Test tubes
• Spectrophotometer, 650 nm
• Waterbath

Procedure:
1. 0.2 mL of BSA working standard (0-1 mg/mL) in 5 test tubes and make up to 1 mL using
distilled water.
2. The test tube with 1 mL distilled water serves as blank.
3. Add 4.5 mL of Reagent I and incubate for 10 minutes at room temperature.
4. After incubation add 0.5 mL of reagent II and incubate for 30 minutes at 50°C.
5. Measure the absorbance at 650 nm and plot the standard graph.
6. Estimate the amount of protein present in the given sample form the standard graph.

LAB MANUAL

 UNIKL MICET 2
CFB 20703 FOOD CHEMISTRY

Study question:

1. The procedure has been widely used in protein biochemistry, however, very few in
determining proteins in food system. Discuss why and how can the Lowry’s’ be applied for
food system?
2. Based on the concentration of the protein obtained from this experiment, discuss any
chemical interference that would be taken into account besides than that of human error?

References:

1. Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein
measurement with the Folin phenol reagent. Journal of biological chemistry, 193(1), 265-
275.

LAB MANUAL