Biochemistry 311 Biochemical methods 2018 Prof Dean Goldring.

Outcomes and objectives.

General: Students should be able to design experiments to isolate, detect and determine
protein, carbohydrate, fat and nucleic acids from a range of tissues and bacteria. Students
should be able to interpret and explain data from such experiments. Students are expected
to be able to interpret a scientific journal article and write experimental data in the format
of a scientific journal article.

1. Design experiments to test pH. Calculate pH based on the Henderson Hasselbalch

equation.
2. Interpret Spectra, draw energy diagrams, interpret data and explain the Beer-Lambert law.
Identify difference spectra and explain their significance.
3. Describe the chemistry and reactions of Fluorescence and chemiluminescence. Outline of
experimental uses of the methods.
4. Describe methods to isolate and separate proteins, carbohydrates, fats and nucleic acids.
Provide reasons and arguments for the choice of starting material.
5. Describe the principles employed in the centrifugation of biological materials. Calculate
the relative centrifugal force experienced by a particle under centrifugation. Explain the
rate of sedimentation during centrifugation. Identify the types of centrifuges and rotors
and explain their specific characteristics and uses.
6. List safety rules for operating a centrifuge. Explain and describe the different types of
separation, differential centrifugation, density gradient.
7. Describe the ingredients of cell media and their role in cell culture. Explain how to use a
haemocytometer and interpret data obtained from its use.
8. Outline the techniques and components of media used for culturing of bacteria.
9. Describe the preparation and extraction of lipids and carbohydrates. Explain the
principles employed in the extraction process and relate principles to the chemical
structure and physical characteristics of lipids and carbohydrate.
10. Describe the principles used in the extraction of Nucleic acids, and relate principles to the
structure of DNA and RNA.
Proteins and detection.
11. Explain the chemical interactions when organic solvents, polyethylene glycol and
ammonium sulphate are used for the separation of proteins.
12. Indicate how carbohydrates, lipids, Nucleic acids, proteins and amino-acids can be
detected and determined in the laboratory. Explain the chemistry of the reactions.
13. Outline the steps in the Southern blot DNA hybridisation technique.
14. When determining protein concentration with the Bradford, Lowry and Goldring
methods, explain the role of acid, alkali and colour in the methods.
Electrophoresis
15. Describe the movement of ions under electrophoretic conditions. Outline the steps to
running polyacrilamide, agarose, and native gels. Relate the gel types to predicted
outcomes and appropriate uses.
16. Describe the influence of sodium dodecyl sulfate (SDS) on proteins in polyacrilamide
gel electrophoresis (PAGE). Employ the method to determine molecular weight of
proteins. Interpret results from such an experiment.
17. Predict the movement of charged proteins in an isoelectric focussing gel. Make choices
regarding voltage, time and gel consistency. Link advantages of 2D gels to IEF and SDS
PAGE.
18. Design an experiment to determine protein presence on nitocellulose (Western blotting).
19. Design an experiment to separate proteins using chromatographic techniques and the
principles of filtration.
20. Explain the movement of proteins in an ion-exchange chromatography matrix. Explain
the influence of pH, charge and ligands on protein/matrix interactions.
21. Understand ligand interaction to describe affinity chromatography.
22. Use hydrophobic interactions to explain hydrophobic chromatography.
23. Design experiments in thin layer (TLC), HPLC, Gas (GLC) chromatography. Explain the
role of temperature, matrix and pressure in the different systems and their operation.
24. Describe radioactive decay and the use of radioisotopes in biochemical studies.
25. Calculate efficiencies of radioactive counting. Determining half life of a radioactive
substance.
26. Detecting and determining radioactive decay.

Literature understanding.
1. Describe, understand and compile the different sections of a Scientific paper e.g. Title,
abstract, key words, author addresses, introduction, materials, methods, results,
discussion, references, acknowledgements.
2. Describe where to obtain appropriate information for conducting a literature review,
experiment or citation of references.
3. Enterpret experimental results on any aspect covered by the module in class or in the
laboratory.

Experimental section.
1. Perform any of the experiments in the laboratory manual.
2. Interpret any of the data generated by any of the experiments from the laboratory manual.
3. Keep accurate records during an experiment.
4. Draw graphs, tables, figures to depict experimental data generated in the laboratory.
5. Write an experiment in the form of a scientific paper. E.G. Title, abstract, key words,
introduction, materials, methods, results, discussion, references, acknowledgements.
6. Be able to calibrate a pH meter, set up a spectrophotometer, weigh reagents, balance and
run samples on a bench top centrifuge.
7. Keep a laboratory note book to “good lab practice standards”.