Isoelectric precipitataion of Proteins : Casein
from milk Milk contains various types of proteins, with
most of them in very small amounts. Traditionally milk proteins were classified into caseins,
whey proteins and minor proteins. Casein is a group name for the dominant class
of proteins in milk. It is responsible for the white, opaque appearance of milk, as it
combines with calcium and phosphorus to form clusters of casein molecules, called micelles.-
The isoelectric point of a protein (I.E.P.) is the PH at which the molecule is electrically
neutral (net charge is zero). As most proteins show minimum solubility at
their isoelectric point, this principle is used to isolate casein by adjusting the PH
of milk to (4.5-4.8) its isoelectric point. As the PH of Milk is much higher than the
isoelectric point of casein to precipitate the casein dilute hydrochloric acid is added
drop by drop to the milk, until the isoelectric point is reached
Apparatus: 100 ml milk, distilled water, 0.2N HCl, burette,
magnetic stirrer, PH meter. Procedure: 100 ml of milk is centrifuged at 4000 rpm
for 20-25 minutes at room temperature. To get the skimmed milk required for the procedure,
remove the cream/fat that settles on the surface of the milk carefully with the help of a spatula.
Transfer the skimmed milk into a 500 ml glass beaker.
Add equal volume of water to the skimmed milk. Now take a 50ml burette which will be used
to add the HCl. Place a magnetic stirrer on the burette stand.
Put a magnetic bar into the beaker containing the milk.
Using the magnetic stirrer, stir well the contents in the beaker.
Take 0.2N HCL and pour it into the burette. Now remove the electrode of the PH meter from
the KCl solution. Wash the electrode using distilled water as
shown. Wipe the electrode using a tissue paper as
shown. Dip the electrode into the beaker containing
milk and check the pH. The pH should be ~6.6.
Now arrange the apparatus as shown. While stirring the milk and add HCL drop by
drop Casein starts precipitating at a pH between
4.8 to 4.6 Wait till the precipitated casein settles
down. Filter out the Casein by pouring it through
a funnel and a filter paper. Wash the precipitated casein first with distilled
water and then with ethanol and lastly with diethyl ether.
Now transfer the cakes of casein deposits from the filter paper into a Petri plate.
Allow the filtered casein to dry and then check the weight.
The weight of the same should be 3.5 gms.