1. Lab Activity2Proteins IUG, 2013 Dr. Tarek M Zaida

3. Background

4. Experiment1: Precipitation of casein at it’s pI • Reagents & Instruments • Casein powder, • solutions of : • 1 N NaOH, • 1 N CH3COOH, • H2Od, • Volumetric flask

5. Procedure 1. Into a 50 ml volumetric flask add 20 ml of water. 2. Add 0.25 g of pure casein, followed by the addition of 5 ml of 1 N NaOH solution. 3. Once casein is dissolved, add 5 ml of 1 N acetic acid solution, then dilute with H2O to 50 ml and mix well. The resulted solution is a 0.1 N casein acetate sodium. 4. Setup a series of 9 test tubes as shown in the table below. 5. In the first test tube put 3.2 ml 1 N CH3COOH, and 6.8 ml H2O and mix thoroughly. 6. In each of the other test tubes (2-9) put 5 ml H2Od. 7. From the test tube 1 transfer 5 ml to the test tube 2, and mix thoroughly.

6. 8. Repeat step 7 for the rest of test tubes (3 - 9). 9. Now to each test tube (1 -9) add 1 ml of the casein acetate sodium solution, and shake the test tubes immediately. 10. Let the samples stand for 30 min, and note the turbidity in the 9 test tubes. 11. Use + and – signs to describe the turbidity in the different test tubes. 12. You should observe the most precipitation in the test tube which has the pH around 4.7 (close to the isoelectric point of casein).

9. Background

12. Casein can be precipitated by: 1. Calcium ions 2. HCl 3. Renin 4. Bacteria

13. Experiment2: Isolation of Casein from cow milk • Reagents • 20 ml milk, • Glacial acetic acid (100%), • Ethanol (95% v/v), • Ether, Thermometer to 100 oc.

14. Procedure 1. Place 20 ml (20 g) of milk into a 125 ml flask and heat at 40 oC in a water bath. 2. Add 5 drops of glacial acetic acid and stir for about 1 min. 3. Filter the resulting mixture through 4 layers of cheesecloth held in a funnel and gently squeeze out most of liquid. 4. Remove the solid (casein and fat) from the cheesecloth, place it into a 100 ml beaker and add 10 ml of 95% ethanol.

15. 5. Stir well to break up the product. Pour off the liquid and add 10 ml of 1:1 ether-ethanol mixture to the solid. Stir well and filter through 4 layers of cheesecloth. 6. Let the solid drain well, then scrape it into a weighed filter paper and let it dry in the air. • Calculate the casein percentage in milk as follows: % Casein =__grams of casein__ x 100 • grams of milk