Experiment No.
2
ISOLATION AND CHARACTERIZATION OF PROTEINS
2.1 Introduction
2.2 Objectives
To isolate casein from skimmed milk/non-fat milk by isoelectric precipitation;
To analyse chemical groups responsible for the color reactions and explain the
principle involved in each test;
To perform acid and base hydrolysis on the isolated proteins
2.3 Isolation of Casein
2.3a Materials and Reagents
Powdered non-fat milk/skimmed milk
10% acetic acid
Thermometer
pH meter/indicator
Funnel and filter paper
Hot plate
2.3b Procedure
1. Place 20.0 g of powdered non-fat milk and 50.0 mL of water into a 100 mL
beaker. Mix well
2. Heat the mixture to about 55˚C (Note: Do not exceed 55˚C. Monitor the
temperature with a thermometer
3. When the mixture has reached 55˚C, add 10% acetic acid dropwise. Stir the
solution gently after every 5 drops
4. Continue adding acetic acid until the pH reaches 4.6
5. Filter off the congealed casein by vacuum of gravity filtration. Dry the casein
residue and calculate the weight % of casein isolated from the powdered milk
2.4 Acid and Base Hydrolysis of Intact Proteins
2.4a Materials and Reagents
Isolated casein and albumin
8N and 16N H2SO4
Cotton plug
Autoclave
Saturated Ba(OH)2
2.4b Procedure for Acid Hydrolysis
1. Add 5 mL 8N H2SO4 to 0.5g isolated protein into a 125 mL Erlenmeyer flask
2. Stopper the flask with cotton plug and cover with aluminum foil then autoclave
(15 psi) for 5 hours.
1. Observe the blackening of the sample after autoclaving. Dilute the hydrolyzate
with 15 mL of distilled water and then transfer the contents to a 250 mL beaker
3. Neutralize the mixture by adding saturated Ba(OH)2. Check the pH using pH
paper.
4. Filter the neutralized mixture. Set aside the filtrate (hydrolyzate) for procedure
2.5
2.4c Alkaline Hydrolysis of Intact Proteins
2. In a 125 mL Erlenmeyer flask, add 3 mL of boiling water and 5 mL saturdated
Ba(OH)2 to 0.5gof the protein isolate
3. Label the flask and plug with cotton plug. Cover with aluminum foil
4. Autoclave the flask at 15 psi for 5 hrs. note the appearance before and after
autoclaving
5. Dilute the hydrolyzate with 15 mL of distilled water and then transfer the
contents to a 250 mL beaker
6. Neutralize the hydrolyzate by adding 1.0 mL of 16 N H2SO4 dropwise. Check the
pH using pH paper.
7. Filter off the precipitate and set aside the filtrate (hyrdolyzate) for procedure 2.5
2.5 Qualitative Color Reactions
2.6a Materials and Reagents
Intact and hydrolyzed protein Conc. HNO3
samples Conc. H2SO4
Biuret reagent 6M NaOH
Millon’s reagent 2% NaOBr
Hopkins-Cole reagent Pb(AOc)2
Sakaguchi reagent Hot Plate
0.1% Ninhydrin solution Beaker
Conc. NaOH solution
2.6b Procedure
1. Sample Preparation. Add 1 mL of distilled water to 0.5g of the intact protein and
0.5 mL of hydrolysed samples.
2. Biuret Test. Add 10 drops Biuret reagent to the sample. Shake the test tube and
note the color change.
3. Ninhydrin Test. Place 6-10 drops of 0.1% Ninhydrin solution into the diluted
samples. Heat the test tube in a boiling water bath. Take note of the appearance
of blue-violet coloration.
4. Xanthoproteic Test. Slowly add 10 drops conc. HNO3 to the diluted samples. Mix
and note the color of the solution. Slowly add 10 drops conc. NaOH. Mix and note
again the color of the solution
5. Millon’s Test. Add 5 drops Millon’s reagent to the diluted samples. Note the
change in color.
6. Hopikins-Cole Test. Slowly add 20 drops Hopkins-Cole reagent to the samples.
Mix well. Incline the test tube and add slowly along the side 20 drops conc.
H2SO4. Do not shake the mixture. Note the color at the interface.
7. Sakaguchi Test. Add 10 drops Sakaguchi reagent to the diluted samples. Mix and
let stand for 3 minutes. Add 3 drops of 2% NaOBr. Mix and note the color
produced.
8. Fohl’s Test. Add 20 drops 6 M NaOH and a few crystals of Pb(OAc)2 to the diluted
samples. Mix well. Heat the mixture in a boiling water bath for 5 minutes. Note
the formation of black precipitate.
2.7 Results and Observation.
1. Isolation of Proteins
Proteins Description
Casein %w/w:____
Albumin
2. Hydrolysis of Intact Proteins
Mode of Hydrolysis Description of Hydrolysate before and
after autoclaving
3. Qualitative Color Reactions
Color Reaction Intact Protein Hydrolyzed Protein Inference
Biuret
Ninhydrin
Xanthoproteic
Millon’s
Hopkins Cole
Sakaguchi
Fohl’s
