Exercise 4

Question 1

exercise 4 title

Answer 1

techniques for protein isolation

Question 2

two techniques in protein precipitation

Answer 2

1. salting out process
2. isoelectric precipitation

Question 3

amount of egg used in isolation of crude ovalubumin

Answer 3

2 egg whites filter through cheesecloth

Question 4

steps in isolation of crude ovalubumin from egg white by ammonium sultae precipitaiton (salting out)

Answer 4

1. 2 egg whites, stir, filter through cheesecloth
2. measure 40 mL
3. add 2.4 mL of 1.7 M acetic acid, slowly, constant stirring
4. strain cheesecloth
5. obtain 30 mL
6. add 7.26 g ammonium sulfate to bring to 40% saturation
7. strain cheesecloth
8. add 3.90 g ammonium sulfate to bring to 60% saturation
9. ice bath 30 min. occasional stirring
10. filter paper

Question 5

steps in isolation casein

Answer 5

1. 5 g powdered non-fat dry milk, 20 mL warm water (38C)
2. warm to 55C
3. add 1.7 M acetic acid slowly (less than 1.2 mL)
4. keep beaker on hot plate until liquid is clear
5. stir precipitated casein and remove
6. filter
7. press solid with spatula
8. place casein in 100 mL beaker
9. add 5 mL of 1:1 ethyl ether and ethanol
10. stir casein in ether, decant, ether, repeat to 5 mL protion of ether
11. filter

Question 6

isoelectric precipitation of casein

Answer 6

1. place 0.1 g casein to 50mL volumetric flask
2. add 25mL distilled (38C)
3. add 5mL 1 M NaOH
4. dissolve casein
5. add 2.9 mL of 1.7 M acetic acid
6. mix, cool, and fill up to 50 mL
7. place 1mL of solution to three test tubes
8. allow to stand 30 min. observe cloudiness

Question 7

three test tubes in isoelectric precipitation of casein

Answer 7

1. 10mL glycine-HCl buffer (pH 2.7)
2. 10mL acetate buffer (pH 4.7)
3. 10mL phosphate buffer (pH6.7)