1. Biochemistry Demonstration Dept. of Biochemistry and Molecular Biology Professor Wu Yaosheng 2012-09

2. Experiment One 1. Basic Demands 2. Basic Operation 3. Spectrophotometry 4. Assay of Protein Concentration (Coomassie Brilliant Blue G-250)

3. 1. Basic Demands To abide the rules in laboratory ◆ Attendance at practicals is compulsory ◆ Always wear a laboratory coat during experiment process ◆ Do not smoke, eat, chew, drink in the lab ◆ Keep your area of bench tidy and organized ◆ When handling hazardous solution or gas, using ventilation cabinet ◆ Experiment reports are required and submitted by the due date ◆ Keep the rules for the students on duty at the end of exp.

4. Experiment report demands Name of the experiment Date of the experiment Principle Protocol Results (phenomenon and data ) Calculate Analysis and discussion

5. 2. Basic Operation Pipeting Automatic pipettes Stir and blend Clean the glass vessel and graduated pipettes after used

7. Pipette with only scale

8. 3. Spectrophotometry Principle: Beer-Lambert law A=kCL Absorbance Constant concentration path length of light

9. Absorbance ( optical density or extinction) Absorbance is measured by the spectrophotometer A = log 10 I0I0 I I 0 ----the intensity of the incident light I0I0 I I ----the intensity of the transmitted light through the solution

10. 3. Spectrophotometry Principle: Beer-Lambert law A=kCL A is proportional to C of the solution 5% CuSO 4 solution, copper sulfate, bluestone

11. (1) To calculate the Conc. of an unknown solution using standard tube Standard solution ： As=KsCsLs (1) Unknown solution ： Au=KuCuLu (2) (1)/(2), As Au = Cs Cu = Au As × Cs

12. (2)To obtain the Conc. of unknown solution by standard curve method TubeBlank 12345 Conc.C1C2C3C4C5C6 Absorb ance A1A2A3A4A5A6 A C 0 X-coordinate Y-coordinate

13. The demands for a standard curve Arrangement in reason Indicate unit for concentration Other information （ the name of the curve, the operator, the date, apparatus or machine used, et al.)

14. The demands for a standard curve Name of the curve Operator Date Apparatus A C 0

15. (2)To obtain the Conc. of unknown solution by standard curve method TubeBlank 12345 unkown Conc.C1C2C3C4C5C6Cu?Cu? Absor bance A1A2A3A4A5A6AuAu A C 0 AuAu CuCu

16. 4. Assay of Protein Concentration (Coomassie Brilliant Blue G-250) The methods for the measurement of protein content in samples To determine the nitrogen content Spectrophotometry Ultraviolet spectrophotometry Visible light spectrophotometry （ various Colorimetric reaction ）

17. Coomassie Brilliant Blue G-250 Aims: To learn how to assay protein conc. Using Coomassie Brillilant Blue G-250 dyeing method Principle: Coomassie Brilliant G-250 + Pr Colour of the solution changed from yellow to blue H+H+ Absorbance peak is at 595 nm

18. Protocol 1. Make a standard curve Reagent123456Unkown Pr standard (0.1mg/ml) 0.10.30.50.70.90Sample 0.1 NaCl (9g/L)0.90.70.50.30.11.00.9 Pr reaction solution 5.0 Corespondi ng Pr conc. (mg/ml) 0.010.030.050.070.090? A 595 0 ( mlL )

19. Coomassie Brilliant Blue G-250 standard curve for protein assay Operator Date Apparatus 0 A 595 C mg/mL C g/L 0.02 0.04 0.06 0.08 0.10 0.1 0.2 0.3 0.4 0.5

20. Protocol 2. Assay of the blood serum specimen (1) Dilute 200 times for the serum (has been done by technician in lab. (2) Manipulate the diluted sample following the method of making standard curve. (3) Find the protein concentration from the standard curve according to A 595.

21. Calculation Blood serum protein conc. (g/L) = data located from the standard curve × 200 0.1 200----diluted times 0.1----the volume of dilution serum sample Normally, reference value extent of serum protein for an adult: 60~80g/L

22. Discussion 1. In which case will the protein conc. in serum decline? 2. Is it correct for your measurement of the serum protein? Why ?