1. Experiment Two 1. Electrophoresis of serum protein (CAME) 2. Assay of casein isoelectric point ( precipitation method) 2012-09

2. 1. Electrophoresis of serum protein (CAME) 【 Aims 】 1. To understand the principle of CAME ( Cellulose Acetate Membrane Electrophoresis ) 2. To learn how to analyze the relative conc. of various proteins in serum

3. 1. Electrophoresis of serum protein (CAME) 【 Principle 】 When placed in an electric field, molecules with a net charge such as proteins, will move towards one electrode or the other, a phenomenon known as electrophoresis. If a protein has more net charges, it will run faster, otherwise reverse. According to this phenomenon, the proteins in serum can be separated into 5 groups, including A, α 1,α 2, β,λglobulin

4. - + + + + + - - - - - + + + + + - - - - 【 Principle 】 The speed of running depends on the amount of net charge and molecular weight of the protein molecule

6. 1. Electrophoresis of serum protein (CAME) 【 Protocol 】 1. Preparation before Electrophoresis (1) Dip the piece of CAM in the buffer(pH=8.6) for ten minutes (2) Put some serum (2~3µl) on the CAM piece by pipette (3) Take the piece of CAM on the Electrophoresis instrument (Caution: the side with serum must be put on (-) electrode)

7. 1. Electrophoresis of serum protein (CAME) 【 Protocol 】 2. Electrophoresis Voltage: about 100~120v Time: 40~60min 3. Dyeing and rinsing ( washing ) When Electrophoresis finish, take out the piece and put it into the protein dyeing for 10~15min then rinsing with the washing solution 1 till its background is cleaned.

8. 1. Electrophoresis of serum protein (CAME) 【 Protocol 】 4. Determination of the amount of proteins To cut each protein strip and blank strip, put them into 5ml of the washing solution 2 respectively, shake up several times, read A 500 after half an hour. T=A+α1+α2+β+ γ

9. 1. Electrophoresis of serum protein (CAME) 【 Reagents 】 1. Barbital buffer (pH8.6) 2. Protein dyeing solution 3. Washing solution1 (C 2 H 5 OH 45ml + CH 3 COOH 5ml + H2O 50ml) 4. Washing solution 2 (0.4mol/L NaOH)

10. 1. Electrophoresis of serum protein (CAME) 【 Caution 】 The end of piece of CAM with serum should be put on the negative polar on the electric field. Take care about the voltage, don’t leave it too high which will damage the electric field because of overheating.

11. 1. Electrophoresis of serum protein (CAME) Table 3 Some data of proteins in serum AlbuminAα1α2βγ pI 4.645.06 5.126.85~7.5 M r (10 thousand )6.95~20309~1530 Ratio ％ of proteins in serum 57~681~65~117~1310~18

12. 1. Electrophoresis of serum protein (CAME) 【 Result 】 【 Discussion 】

13. 2. Assay of casein isoelectric point ( precipition method)

14. Aims To strengthen the comprehension of the protein ionization and pI To learn how to assay protein pI

15. Principle When a protein is at its pI, its net charge is zero and hence its solubility become minimum. According to this, we can place casein in a series of solutions whose pH are different. By observation of the extent of the precipitation, the protein pI will be obtained

16. pH=pI +OH - pH>pI +H + +OH - +H + pH<pI ZwitterionPositively charged ion Negatively charged ion Pr

17. Protocol Tube No.12345 H2OH2O2.43.01.53.5 1.00M HAc1.6 0.10M HAc4.01.0 0.01M HAc2.50.5 casein1.0 Final pH3.54.14.75.35.9 Precipitation Observe precipitation, using “ + ” 、 “ ++ ” “ +++ ” “ ++++ ” to show the extent of the precipitation (ml)

18. clearturbiditywadding deposition turbidity deposition clear deposition -++++++++++ Phenomenon