EXPERIMENT NO.

ANALYSIS OF PROTEINS

OBJECTIVE:

At the end of the experiment, the learners should be able to:

1. isolate casein from commercial milk;

2. test the isolated casein for its protein properties using chemical tests;

3. observe selected characteristic color reactions of amino acids and

proteins; and

4. predict the typical chemical groups present in proteins through

chemical tests.

MATERIALS/REAGENTS

Erlenmeyer flask 250mL Bunsen burner tripod

Water bath filter paper/cheese cloth funnel

Test tubes with rack test tube holder litmus paper

Lead acetate paper medicine dropper

Evaporated milk 1% egg albumin glacial CH3COOH

1M NaC2H3O2 ethanol ether

0.1M NaOH 10% NaH dil. CuSO4

0.5% sulfanilic acid 0.5% HNO3 10% Na2CO3

Hopkin’s cole reagent Millon’s reagent 0.1M HCl

Ninhydrin reagent 3M NaOH conc. HNO3

Conc. HCl 5% Pb(C2H3O2)2 5% HgCl2

5% FeCl3

PROCEDURE:

1. Isolation of Casein from milk

1. Place 50g of milk into 250mL Erlenmeyer flask and heat the flask in a
water bath. Stir constantly the solution using a stirring rod. When the
temperature reach 40oC, remove the flask from the water bath. Add 10 drops of
glacial acetic acid while constantly stirring.

2. Note the formation of precipitate. Filter using cheesecloth and collect

the precipitate using spatula into an empty beaker.

3. Add 25mL of 95% ethanol to the precipitate. Stir for 5 minutes and
decant. Filter and discard the liquid and place the residue in an empty beaker.
Add 25mL of 1:1 mixture of ether-ethanol. Stir for another 5 minutes and
collect the solid by filtration. The solid is the casein.

Caution: Ether and ethanol are both flammable liquids. Perform

under the hood.

4. Spread the casein on a clean dry filter paper and let it dry. Weigh the
dried casein and calculate the percentage recovery. Record.

2. Preparation of Casein Solution

1. Dissolve the casein (obtained from isolation) with distilled water. The
amount of distilled water will depend on the amount of casein. If you have
recovered about a spoonful of casein, you may dissolve it with 10mL of distilled
water. Use 20mL casein for the following analysis.

3. Test for Proteins

For every test, prepare five test tubes 2mL of the following solutions:

Test tube 1- casein solution

Test tube 2- 2% glycine

Test tube 3- 2% gelatin

Test tube 4- 2% albumin

Test tube 5- 1% tyrosine

A. Biuret Test

To each test tube, add 2mL of Biuret reagent and mix thoroughly. Wait
for 2 minutes and examine each tube and note the change in color. Record
your observation.
 B. Hopkins-Cole Test

To each test tube, add 0.5mL of Hopkins-Cole reagent. Shake. Add

dropwise conc. H2SO4 until two layers become visible. Note the color at the
junction of the 2 liquids.

C. Millon’s Test

To each test tube, add 5 drops of Millon’s reagent. Heat the mixture in a
water bath for 3 minutes. Note the results.

D. Ninhydrin Test

To each test tube, put 1mL of 0.2% Ninhydrin solution and heat for
several minutes in boiling water bath. Record your observations.

E. Sulfur Test

To each tube, place 1mL of 3M NaOH. Heat gently in water bath for 15
minutes. Cool and acidify by adding 3M HCl dropwise until blue litmus paper
turns to red. Place a moist lead acetate paper over the mouth of each test tube.
Boil the contents. Record your observation.

F. Xanthoproteic Test

Caution: Perform the experiment under the hood. Be very careful in

handling conc. HNO3.

To each test tube, carefully add 10 drops of conc. HNO3 while swirling.
Heat the mixture in a water bath for 1 minute. Note the change in color.

4. Denaturation Tests

A. Place 2mL of casein suspension in a test tube and heat until the
solution boils. Observe the appearance of the casein suspension after heating.

B. Get a second test tube and place 2mL of casein suspension along with
8mL of 95% ethanol. Shake well and note the result.

C. Prepare three test tubes and label them as test tubes 1, 2 and 3. Place
1mL of casein suspension in each test tube and add the following solutions
dropwise:

- To test tube 1, add a few drops of 5% lead acetate

- To test tube 2 and 3, add a few drops of 5% mercuric chloride and 5%

ferric chloride respectively.
Note the test tube where precipitate is formed.

Note: Discard all the solutions down the drain with plenty of running
water. Pour the mercury solution into designated chemical wastes
container.
Name:___________________________ Date:_____________

Course/Year:______________________ Score:_____________

REPORT SHEET

ANALYSIS OF PROTEINS

DATA AND OBSERVATIONS

A. Calculate the percentage of casein in the milk

% casein= weight of solid casein/ 50g of milk x 100

Computation:

B. Test for Proteins

1. Biuret Test

Solutions Observation/Positive Color Reaction

Test tube 1
Casein suspension

Test tube 2
2% glycine

Test tube 3
2% gelatin

Test tube 4
2% albumin

Test tube 5
1% tyrosine
2. Hopkins-Cole Test

Solutions Observation/Positive Color Reaction

Test tube 1
Casein suspension

Test tube 2
2% glycine

Test tube 3
2% gelatin

Test tube 4
2% albumin

Test tube 5
1% tyrosine

3. Millon’s Test

Solutions Observation/Positive Color Reaction

Test tube 1
Casein suspension

Test tube 2
2% glycine

Test tube 3
2% gelatin

Test tube 4
2% albumin

Test tube 5
1% tyrosine
4. Ninhydrin Test

Solutions Observation/Positive Color Reaction

Test tube 1
Casein suspension

Test tube 2
2% glycine

Test tube 3
2% gelatin

Test tube 4
2% albumin

Test tube 5
1% tyrosine

5. Sulfur Tests

Solutions Observation/Positive Color Reaction

Test tube 1
Casein suspension

Test tube 2
2% glycine

Test tube 3
2% gelatin

Test tube 4
2% albumin

Test tube 5
1% tyrosine
6. Xanthoproteic Test

Solutions Observation/Positive Color Reaction

Test tube 1
Casein suspension

Test tube 2
2% glycine

Test tube 3
2% gelatin

Test tube 4
2% albumin

Test tube 5
1% tyrosine

C. Denaturation Tests

Solutions Observation/Appearance after Denaturation

Casein suspension

Casein suspension +
ethanol

Test tube 1
Casein suspension +
lead acetate

Test tube 2
Casein suspension +
HgCl2

Test tube 3
Casein suspension +
FeCl3

Questions:

1. Explain why casein precipitates when acetic acid is added to it?

2. Cite five examples of denaturation of proteins encountered in everyday

life.

3. Why is milk or raw egg used as antidote in cases of heavy metal ion
poisoning?