1. BIOCHEMISTRY EXPERIMENT SSheng Zhao ( 赵晟 ) WWeb for lecture slides ： http://teaching.ewindup.info EEmail/QQ /MSN/gchat: shengzhao@seu.edu.cn or windupzs@gmail.com MMobile:18551669724 or 13675130010 Principle + Practice Logic analysis

2. Introduction  From public criticism: some schools teach current students by past knowledge to face the future world.  How to face the future ？： deal with the real world.  How to deal with the real world? Data and facts Professor John Ross: fighting with rumors using the data and facts on Sina Weibo, the Chinese Twitter. As foreign students: conduct ex periments carefully and no cheat on exam.  Principle + practicelogic analysis

3. Introduction Biochemistry:  Biology  Chemistry  Molecular level  Biologic structures and functions. Major methods:  Laboratory assays  Interdisciplinary

4. Examination for Biochemistry class 1.Class participation: 20% Lecture and experimental training, logic analysis, and experiment reports. 2.Experiential exam: 10% Perform a specified experiment independently. 3.Exam on biochemistry theory (Lecture): 70%

5. Preview. Wear the white coat. Be on time. Reagents back to place after usage. Welcome for any question. Finish report in class. Clean the room after class. Requirement

6. 1.Title; 2.Principle ； 3.Procedures(simple) ； 4.Results and analysis; 5.Conclusions. Report (Weibo/Twitter style)

7. Using the pipette

8. Adjustable ： 0.5-2ΜL, 2-20ΜL, 20-200ΜL, 100-1000ΜL, 1000- 5000ΜL

9. Usage ： 1.Select the right pipette and tips 2.Holding of pipette 3.Sampling 4.expelling of liquid 5.Discharge the pipette tip

10. Class syllabus by logic Protein quantification: Folin phenol (Lowry) assay Ion exchange chromatography for the mixed amino acids Transamination Protein purification Gel filtration chromatography Sodium dodecyl sulfate-Polyacrylamide gel electrophoresis (SDS-PAGE) Serum triglyceride (TG) measurement Plasmid DNA extraction, restriction enzyme digestion, and agarose gel electrophoresis Enzyme Km Serum glutamic-pyruvic transaminase (ALT) measurement (Exam!) Protein analysis Nucleic acid analysis Protein function Lipid analysis

11. Class syllabus by real experiments 1.Introduction and Protein quantification: Folin phenol (Lowry) assay 2.Protein purification: Gel filtration chromatography 3.Plasmid DNA extraction, restriction enzyme digestion, and agarose gel electrophoresis 4.Enzyme Km 5.Transamination 6.Sodium dodecyl sulfate-Polyacrylamide gel electrophoresis (SDS- PAGE) 7.Ion exchange chromatography for the mixed amino acids 8.Serum triglyceride (TG) measurement 9.Serum glutamic-pyruvic transaminase (ALT) measurement (Exam!)

12. PROTEIN QUANTIFICATION: FOLIN PHENOL (LOWRY) METHOD Folin phenol (Lowry) assay Usage of spectrophotometer Plotting of standard curve

13. Common methods for protein quantification 1.UV Spectrophotometry 2.Lowry assay 3.Coomassie brilliant blue -G250

14. Folin phenol (Lowry) assay Step 1 Under alkaline condition protein reacts with Cu and forms complex Dark blue A 650 In a certain range the strength of the color is proportional to protein concentration. Complex reduce the phenol reagents Step 2

15. Spectrophotometry Lambert―Beer law ： In optics, the Lambert―Beer law relates the absorption of light to the properties of the material through which the light is travelling.

16. Advantage Simple and sensitive (25-250 μg) Disadvantage Time consuming ， interfered by a wide variety of chemicals Sensitivity: 5 µ g/ml ， Measurement range: 25~250 µg/ml

17. 1.Use the standard solution. 2.Measure the absorption at a certain wave length. 3.Plot the standard curve: absorbance as ordinate and the protein as horizontal coordinate. Standard curve

18. Cross the zero point A is between 0.05 ～ 1.0 650nm 0.125 0.25 0.375 0.5 0.625 mg

19. Procedure （ 7 tubes ） 1234567 Standard solution —0.20.40.60.81.0— Measured sample ——————1.0 H2O1.00.80.60.40.2—— A5555555 Mixing, 10min at RT B0.5 Mixing, 30min, 650nm Absorption Protein content (μg) A 650nm

20. REPORT Title Principle Procedure Results and analysis tube1234567 A Standard curve