Source: Biochemistry and Molecular Biology Laboratory Guide, Shao Xueling et al, Wuhan University Press, 2003.

Operation method

isoelectric point precipitation method

Principle

The main protein in cow's milk is casein, with a content of about 35 g/L. Casein is a mixture of a number of phosphoproteins, with an isoelectric point of 4.7. Using the principle that solubility is lowest at the isoelectric point, casein precipitates when the pH of cow's milk is adjusted to 4.7. The precipitate is washed with ethanol to remove the lipid impurities and pure casein is obtained.

Materials and Instruments

Buttermilk
Ethanol Anhydrous ether Acetic acid Sodium acetate Ethanol Ethyl ether
Centrifuge Filtering device Precision pH paper or acidometer Electric stove Beaker Thermometer

Move

1. 100 ml of buttermilk was put into a 500 ml beaker, heated to 40 ℃, and 100 ml of acetic acid-sodium acetate buffer preheated to 40 ℃, pH 4.7 was added slowly with stirring. pH was adjusted to 4.7 with a precision pH paper or an acidimeter. the above suspension was cooled down to room temperature, and then centrifuged (3,000 r/min) for 10 min, the supernatant was discarded or filtered through 4 layers of gauze to obtain the crude casein product. Casein crude product.

2. Wash the precipitate with a small amount of water for 3 times, centrifuge (3000 r/min) for 10 min, and discard the supernatant.

3. Add 30 ml of ethanol to the precipitate, stir for a few moments, transfer all the suspension to a Büchner funnel for filtration, wash the precipitate with a mixture of ethanol and ether for 2 times, and finally wash the precipitate with ether for 2 times, and then pump it dry.

4. Remove the precipitate, spread it out on a surface dish to remove the ether, and dry to obtain the casein pure product. Weigh and calculate the percent content.

Common Problems

1 . Why does casein precipitate at the isoelectric point pH?

2 . Why can protein precipitates be washed with organic solvents?

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