Experiment No.

4
Isolation and Qualitative Tests of Proteins

I. Objectives
1. To isolate the protein casein in a skimmed milk through isoelectric precipitation
2. To test for specific chemical groups in the protein casein

II. Introduction

Proteins are large organic compounds made of amino acids arranged in a linear chain and
joined together by peptide bonds between the carboxyl and amino groups of adjacent amino acid
residues.

To perform in vitro analysis, a protein must be purified from other molecular components.
A mixture can be purified using ultracentrifugation, precipitation, chromatography, and
electrophoresis.

Several qualitative color reactions have been devised f or the detection of proteins.
These tests detect the presence of specific chemical groups in the protein.

III. Materials

A. Equipment

Litmus paper
(2) 250-mL Beaker, (1) buret, (2) 5-mL pipet, Filter paper
pH meter or pH indicator (5)-10-mL Test tubes

Hot Plate

B. Reagents
0.1M HCl
Skimmed milk (ready-to-drink, one per class)
10% NaOH
Hopkins-Cole reagent
0.02% Naphthol solution 2% NaOBr
Millon’s reagent (freshly prepared)
(freshly prepared) Conc. H2SO4
0.1% ninhydrin solution
Conc. HNO3
0.01M CuSO4
Conc. NaOH
2.5M NaOH
0.1 M glycine
0.1 M tyrosine
0.1 M tryptophan
0.1 M arginine
0.1 M cysteine

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IV. Procedure
A. Isoelectric Precipitation of Casein
1. Place 25 mL of skimmed milk in a 250-mL beaker.
2. Add 0.1 M HCl to the milk sample slowly using a dropper The milk sample must be stirred
constantly throughout the process. Stop adding the acid when the pH of the solution reaches a value of
4.6 (use pH meter or a pH paper).
3. Allow the suspension to stand until the precipitated casein settles.
4. Decant and discard the supernatant.
5. Resuspend the precipitate in 10 mL distilled water, then add 0.1 M NaOH dropwise to the suspension
until its pH becomes neutral. Finally, add 40 mL distilled water to the suspension.
6. Divide the casein suspension into two equal portions (approximately 25 mL each). Let the casein in one
25-mL portion settle, then separate it from the supernatant by decantation. Dry the casein by placing it
on a pre-weighed filter paper. Place the casein and the filter paper in an oven set to 100°C. Use the other
25-mL portion for the qualitative tests in part B.
7. Determine the overall weight of the casein that you obtained.

B. Qualitative Tests
1. Biuret Test
a. Add 1 mL of 2.5 M NaOH to 1.5 mL of the sample (casein suspension, 10% albumin, 0.1 M
alanine).
b. Add a drop of 0.01 M CuSO4 solution, then mix. If no definite color develops, add another drop or
two of the CuSO4 solution. Record your observations.
2. Ninhydrin Test
a. Add 0.5 mL of a 0.1% ninhydrin solution to 1.5 mL of the sample (casein suspension, 10%
albumin, 0.1 M alanine).
b. Heat to boiling. Record your observations.
3. Xanthoproteic Test
a. Add 1mL of concentrated HNO3 slowly to 1.5 mL of the sample (casein suspension, 10% albumin, 0.1
M alanine, 0.1 M tyrosine). Caution: HNO3 is very corrosive. Handle it with care.
b. Heat gently.
c. Cool slowly with flowing water.
d. Add concentrated NaOH slowly (drop by drop) until the solution is alkaline. Use litmus paper.
Record your observations.
4. Millon’s Test
a. Add 5 drops of fresh Millon’s reagent to 1.5 mL of the sample (casein suspension, 10% albumin,
0.1 M alanine, 0.1 M tyrosine).
b. Carefully heat the mixture. Record your observations.
5. Hopkins-Cole Test
a. Add 2 mL of Hopkins-Cole reagent to 1.5 mL of the sample (casein suspension, 10% albumin, 0.1
M alanine, 0.1 M tryptophan), then mix.
b. Incline the tube and add slowly 2.5 mL of concentrated H2SO4 down the side of the tube so that two
layers will form. Record your observations. Caution: H2SO4 is very corrosive and releases heat
when added to the reaction mixture. Handle it with care.
6. Sakaguchi Test
a. Add 1 mL of 10% NaOH and 1 mL of 0.02% naphthol solution to 1.5 mL of cold sample (casein
suspension, 10% albumin, 0.1 M alanine, 0.1 M arginine), then mix.
b. After 3 minutes, add 2-4 drops of freshly prepared 2% NaOBr. Record your observations.
7. Nitroprusside Test
a. Add 10 drops of 3 M NaOH to 10 drops of the sample (casein suspension, 10% albumin, 0.1 M
alanine, 0.1 M cysteine), then mix.
b. Add 0.25 mL 2% nitroprusside solution. Record your observations.
8. Fohl’s Test
a. Add 5 drops 30% NaOH and 5 drops 5% lead acetate to 1.5 mL of the sample (casein suspension,
10% albumin, 0.1 M alanine, 0.1 M cysteine), then mix.
b. Place the tube in a boiling water bath. Record your observations.
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 Experiment No. 4
Isolation and Qualitative Tests of Proteins

Group Leader: Date Performed:

Date Submitted:
Group Members:

Year and Section:

I. Isolation of Protein

Total weight of casein obtained:

Physical appearance:

II. Qualitative Tests for Proteins

QUALITATIVE OBSERVATIONS
TEST

Biuret Test

Ninhydrin Test

Xanthoproteic Test

Millon’s Test

Hopkins-Cole’s Test

Sakaguchi’s Test

Nitroprusside Test

Fohl’s Test
Questions:

1. What is meant by the isoelectric pH of a protein?

2. At what pH is a protein least soluble? Why?

3. What type of chemical group is present in all proteins?

4. Give the principle involved and the chemical structure responsible for the positive results in
Biuret, ninhydrin, xanthoproteic, Millon’s, Hopkins-Cole’s, Sakaguchi’s, nitroprusside, and
Fohl’s test.