BIOCHEMISTRY LABORATORY (CHM 527)
Grading:
Laboratories: 100 points
Final examination: 50 points
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Note: the material to be handed in for the labs is shown in italics!
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WEEK 1 (CHAPTER 1- disregard appendix 1-1)
1. Introduction to the laboratory and check-in
2. Safety
3. Orientation of the laboratory
Objectives:
1. What are the important safety rules one has to follow when working in a biochemistry lab?
2. What are the main safety equipment in a typical laboratory?
3. What is OSHA?
4. What are the main requirements of the Federal Hazard Communication Hazard?
5. What is MSDS?
6. What should a Chemical Hygiene Plan contain?
7. What is a positive and negative control?
8. What is the difference between accuracy and precision?
9. What is the difference between precision and reproducibility?
10. How is precision often expressed as?
11. What are the various pipettes and how are they different?
12. What are the main features of a pH meter?
13. What are the main components of a spectrophotometer (eg. Spec 20)?
14. What are some important precautions to take when using a centrifuge?
15. What is the relationship between RCF and RPM?
16.What are the main precautions in washing glassware?
17. Calculations on solutions and dilutions pp.34-36
18. Calculations on the Henderson-Hasselbalch equation.
WEEK 2 (CHAPTERS 2 & 3)
1. Experiment 2-1 p70
i. Prepare 100 mL of 0.2M Tris-HCl, pH 8.0
ii. Prepare 10 mL 0.1 mM PNP in deionized water
iii. Perform experiment 2-1 p.70
What to turn in ? (10 points)
1. Hand in plot of A vs wavelength (from this plot, find the wavelength of maximum absorbance)
2. Determine the extinction coefficient of PNP.
WEEK 3
2. Experiment 3-1; Bradford assay for protein concentration
i. Serial dilute the 2 mg/mL BSA to give ~ 1 mL each of 1, 0.5 and 0.25 mg/mL protein.
ii. Using the standards above, make a standard curve and assay the unknown sample by the Bradford method.
What to turn in ? (10 points)
1. Calibration curve done on a PC
2. Protein concentration of unknown = ___ mg/mL (show observations and calculations)
3. Question: You prepared a calibration curve for a method and determined it was linear from 1-10 mg/ml
protein concentration. You then analyzed an unknown protein sample and determined its value to be 14 mg/mL.
Is this value accurate? What would you do to get an accurate value?
Objectives:
1. What are some of the methods commonly used for measuring protein concentrations in biological samples?
2. What is the quickest method for protein quantitation for pure protein samples ? (hint: no reagent has to be added to the protein
sample in this method)
3. Why do most proteins absorb at 280 nm ?
4. In the absorbance 280 method of protein quantitation, how would you correct for nucleic acid presence in your sample?
5. A serum sample has [protein] = 8 g / dL and consist of various biomolecules besides proteins. Whish method would you use
to measure its protein concentration and why - Biuret. Lowry, BCA, or Bradford?
6. A sample has [protein] = 0.5 mg/mL and has ammonium sulfate, urea, and lipid contamination. Which method would you use
for protein quantitation - Biuret, BCA or Bradford?
7. What is the dye used in the Bradford method?
8. What is the principle of the Biuret method?
9. What metal ions are important in the Biuret, Lowry and BCA methods ?
10. Why is the wavelengths for the Biuret, Lowry, BCA and Bradford methods selected as 550, 750, 560 and 595 nm
respectively ? (hint: look at your results of PNP absorbtion spectrum)
WEEK 4(CHAPTER 4)
1. Lecture on separation techniques Ð chromatography
DO THE MINICOURSE "Some important techniques" Protein separation & analysis in the Lehninger CD while waiting.
2. Pour gel-filtration columns (keep them in the cold room if time runs out).
3. Perform experiment 4-1 p117 (HANDOUT GIVEN IN CLASS)
Objectives:
1. What is Stoke's radius?
2. What chemical is used to measure the void volume of a gel filtration column?
3. What factors cause band broadening in gel filtration?
4. What is the mobile phase in your experiment?
5. Can you use size exclusion vhromatography to determine molecular weight of a protein?
6. The volume of a bed was 20 mL - what would be the volume of the sample applied to the column?
7. In a chromatographic separation of an enzyme, the crude enzyme mixture was applied to a column containing
matrix on to which was immobilized substrate of the enzyme. What is the name of this kind of chromatography?
8. In anion exchange chromatography the stationary phase has a ________ charge (positive or negative?) while in
cation exchange chromatography, the stationary phase bears a ____ charge.
9. What is the isoelectric pH of a protein?
10. What is the chromatography in which the stationary phase is hydrophobic, the sample is applied in a highly
polar buffer and the mobile phase polarity changes (gradient)?
What to turn in ? (10 points)
1. Hand in observation table given out in class.
WEEK 5 (CHAPTER 6)
1. Lecture / Demonstration on protein purification.
WEEK 6 (CHAPTER 7)
1. Perform experiment 7-1 (first day)
2. You may have to come in to the lab for a few minutes to change dialysis solution before next lab period.
3. REMEMBER TO KEEP ASIDE IN THE FREEZER 0.5 mL stage 1 material for enzyme and protein assay.
WEEK 7
1. Perform experiment 7-1 ( Second day)
2. REMEMBER TO KEEP ASIDE IN THE FREEZER 0.6 mL stage 2 material for enzyme and protein assay
WEEK 8 (CHAPTER 5)
1. Lecture on electrophoresis
2. Prepare SDS-PAGE gels and store gels in cold room
Objectives:
1. What factors determine the relative mobility of a protein during electrophoresis?
2. What is the relationship between net charge of a protein and its pI?
3.Most often electrophoresis is done at pH > 8. If a protein has a pI<8, which electrode will it migrate to during electrophoresis,
the cathode (negative electrode) or anode (positive electrode)?
4. The charge to mass ratio of two proteins A and B are the same. The molecular weight of A is twice than that of B. Which
protein will migrate faster during electrophoresis and why?
5. The cross linking agent in PAGE is _________ ?
6. What % acrylamide separation gels would you use if you want to separate proteins of mw <15,000?
7. What is a gradient gel?
8. What kind of electrophoresis is best suited for measuring the mw of an unknown protein?
9. What is the function of SDS?
10. Which has a higher pH, the stacking or resolving gel?
11. What is the function of the stacking gel?
WEEK 9
1. Obtain stage 1,2 and 3 material and run SDS-PAGE electrophoresis as in experiment 5-1. You will have to make arrangements to transfer gel from staining solution to destaining solution (leave in 10/10 MeOH/acetic acid solution) until next lab period.
2. Prepare non-denaturing PAGE gels as in experiment 5-2
WEEK 10
1. Obtain stage 1,2 and 3 material and perform non-denaturing gel electrophoresis as in experiment 5-2.
WEEK 11
1. Obtain stage 1,2 and 3 material and perform enzyme ( p.191-193) and protein assays (use Bradford reagent)
What to turn in ? (50 points) Due week 14
1. Hand-in REPORT :
Introduction, Materials & Methods; Results & Discussion with necessary
graphs, photographs etc. labelled and neatly organized and purification table as shown on p. 172; Conclusion
Due on week 14.
WEEK 12
Catch-up week
WEEK 13 (CHAPTER 8)
1. Determine K m and V max for alkaline phosphatase experiment 8-1.
What to turn in ? (10 points)
1. Hand-in LB plot with necessary calculations
WEEK 14 (CHAPTER 11)
1. DNA digestion with restriction endonucleases and electrophoresis.
2. What to turn in ? (10 points)
1. Hand-in photograph of gel with lanes labelled and brief conclusion of your results.
WEEK 15 (CHAPTER 13)
1. Using PC and the internet for biochemical research.
Examination Week
1. Final examination Ð50 points and check-out