Practical No –

Aim – Isolation of protein by adjusting Isoelectric point.

Principle –

The pH at which there is zero net charge and no migration in an electric field is known as
Isoelectric point. Casein is the main protein found in milk and is present at a concentration of
35 gm/lit. It is a heterogeneous mixture of phosphorous containing protein and not a single
compound. Most protein show a minimum solubility at their Isoelectric point and this
principle is use to isolate casein by adjusting the pH of milk to 4.8, its Isoelectric point.
Casein is insoluble in ethanol and this property is used to remove unwanted fat from
precipitation.

Chemicals & Equipments –

1. Milk (100ml).
2. Ethanol.
3. Ether.
4. Thermometer.
5. Muslin cloth.
6. pH meter.
7. Sodium acetate buffer.
8. Whatmans filter paper.
9. Watch glass.

Procedure-

1. 100 ml of milk was taken in 500 ml of beaker and kept at 40ᵒC in water bath for 10
minutes, also warm 100 ml acetate buffer.
2. Burette filled with sodium acetate buffer was added slowly in milk with constant
stirring.
3. The final pH of the mixture was maintained about 4.8 and checked still for further 10
minutes.
 4. Suspension was filtered using muslin cloth to separate the precipitate.
5. Precipitate was first washed with 30 ml of 100% ethanol (15 ml of ether) and filtered.
6. The precipitate was removed on watch glass and made to dry.

Result-

1. The weight of casein /100 ml =

2. The weight of casein/litre=
3. Standard Casein Value=