10007122Protein Bioseparation Methods
Course Information
Description
Introduces the general strategies commonly used to purify proteins. Specific methods include determining specific activities for enzymes, extraction of proteins from bacterial cells, salting out, dialysis, ion exchange chromatography and polyacrylamide gel electrophoresis.
Total Credits
3
Course Competencies
  1. Identify and explain commercial applications of protein purification methodology.
    Assessment Strategies
    through the completion of written assignments.
    Criteria
    you find examples of commercial protein separations in articles.
    you explain the significance of the products.

  2. Outline a strategy for a protein purification from memory.
    Assessment Strategies
    through written examinations.
    Criteria
    your strategy includes extraction method, low resolution technique, high resolution technique and assay.
    your strategy is consistent with guidelines in text and references.

  3. Identify and explain physical and chemical properties of proteins.
    Assessment Strategies
    through the completion of written assignments.
    through written examinations.
    Criteria
    you relate the physical and chemical properties of a protein to separation methods.

  4. Design a separation strategy based on application of knowledge in 1.3 above.
    Assessment Strategies
    through the completion of written assignments.
    through written examinations.
    Criteria
    you are able to use simulated data from hypothetical protein to design separation strategy.

  5. Explain protein extraction from plant, animal and bacterial cells.
    Assessment Strategies
    through the completion of written assignments.
    through written examinations.
    Criteria
    your methods are explained according to references.

  6. Write a flowchart outlining the extraction of beta galactosidase from E. coli.
    Assessment Strategies
    through the completion of written assignments.
    through written examinations.
    Criteria
    your flow chart includes all steps in lab manual and indicates where samples are removed, volumes determined and assays performed.

  7. Grow and collect E. coli cells on shaker.
    Assessment Strategies
    through the completion of laboratory assignments.
    through the completion of written documentation and explanation of laboratory work.
    through the completion of written explanations of laboratory data.
    through completion of an oral report to class.
    Criteria
    with a pellet of at least 0.5 grams.

  8. Assay supernatant for protein.
    Assessment Strategies
    through the completion of laboratory assignments.
    through the completion of written documentation and explanation of laboratory work.
    through the completion of written explanations of laboratory data.
    through completion of an oral report to class.
    Criteria
    straight line on standard curve; assay result falling on standard curve from sample.

  9. Assay supernatant for beta galactosidase activity.
    Assessment Strategies
    through the completion of laboratory assignments.
    through the completion of written documentation and explanation of laboratory work.
    through the completion of written explanations of laboratory data.
    through completion of an oral report to class.
    Criteria
    by discernible yellow color in sample tube within 10 minutes.

  10. Calculate specific activity of supernatant.
    Assessment Strategies
    through the completion of written assignments.
    through written examinations.
    through the completion of written documentation and explanation of laboratory work.
    through the completion of written explanations of laboratory data.
    through completion of an oral report to class.
    Criteria
    your calculations are correct.
    your results are between 1,000 and 20,000 units/mg.

  11. Keep accurate records of findings.
    Assessment Strategies
    through the completion of laboratory assignments.
    through the completion of written documentation and explanation of laboratory work.
    through the completion of written explanations of laboratory data.
    through completion of an oral report to class.

  12. Describe strategy to precipitate protein.
    Assessment Strategies
    through the completion of written assignments.
    through written examinations.
    Criteria
    your strategy is consistent with textbook guidelines.

  13. Precipitate beta galactosidase from supernatant above.
    Assessment Strategies
    through the completion of laboratory assignments.
    through the completion of written documentation and explanation of laboratory work.
    through the completion of written explanations of laboratory data.
    through completion of an oral report to class.
    Criteria
    your activity in redissolved pellet using assays learned in competency 2.4.

  14. Describe how to desalt a sample and change buffers.
    Assessment Strategies
    through the completion of written assignments.
    through written examinations.
    Criteria
    your description includes all steps of desalting.

  15. Perform dialysis.
    Assessment Strategies
    through the completion of laboratory assignments.
    through the completion of written documentation and explanation of laboratory work.
    through the completion of written explanations of laboratory data.
    through completion of an oral report to class.
    Criteria
    you dialysate with pH of proper buffer, proper conductivity and activity as assayed using methods learned in competency 2.4.

  16. Explain principles of chromatographic separation.
    Assessment Strategies
    through the completion of written assignments.
    through written examinations.
    Criteria
    your explanation is correct according to checklist.

  17. Explain cationic and anionic exchangers.
    Assessment Strategies
    through the completion of written assignments.
    through written examinations.
    Criteria
    your explanation is correct and distinguishes between types of exchangers.
    your explanation discusses pH and charge.

  18. Set up chromatography column with DEAE gel.
    Assessment Strategies
    through the completion of laboratory assignments.
    through the completion of written documentation and explanation of laboratory work.
    through the completion of written explanations of laboratory data.
    through completion of an oral report to class.
    Criteria
    according to manual instructions, all parts are in correct orientation; no bubbles in connecting tube. (Diagram and instructions are in lab manual and Pharmacia booklet.)

  19. Run sample on chromatography column.
    Assessment Strategies
    through the completion of laboratory assignments.
    through the completion of written documentation and explanation of laboratory work.
    through the completion of written explanations of laboratory data.
    through completion of an oral report to class.
    Criteria
    collected fractions.

  20. Graph data from column fractions.
    Assessment Strategies
    through the completion of laboratory assignments.
    through the completion of written documentation and explanation of laboratory work.
    through the completion of written explanations of laboratory data.
    through completion of an oral report to class.
    Criteria
    your graph is similar to that instructor obtained using the assays learned in competency 2.4.

  21. Using centricon apparatus, concentrate sample pooled from column fractions.
    Assessment Strategies
    through the completion of written assignments.
    through the completion of written documentation and explanation of laboratory work.
    through the completion of written explanations of laboratory data.
    through completion of an oral report to class.
    Criteria
    you produce concentrated sample.

  22. Write a short explanation of theory of polyacrylamide gel electrophoresis and isoelectric focusing.
    Assessment Strategies
    through the completion of written assignments.
    through written examinations.
    Criteria
    you explain differences between the two methods.

  23. Cast a PAGE gel.
    Assessment Strategies
    through the completion of laboratory assignments.
    through the completion of written documentation and explanation of laboratory work.
    through the completion of written explanations of laboratory data.
    through completion of an oral report to class.
    Criteria
    your gel is completed.

  24. Run samples on PAGE.
    Assessment Strategies
    through the completion of laboratory assignments.
    through the completion of written documentation and explanation of laboratory work.
    through the completion of written explanations of laboratory data.
    through completion of an oral report to class.
    Criteria
    your gel is completed.
    mw markers with correct spacing.
    effective protein separation.

  25. Perform a Western Blot.
    Assessment Strategies
    through the completion of laboratory assignments.
    through the completion of written documentation and explanation of laboratory work.
    through the completion of written explanations of laboratory data.
    through completion of an oral report to class.
    Criteria
    you perform blot and B galactosidase band is present.

  26. Cast an Isoelectric Focusing (IEF) Gel.
    Assessment Strategies
    through the completion of laboratory assignments.
    through the completion of written documentation and explanation of laboratory work.
    through the completion of written explanations of laboratory data.
    through completion of an oral report to class.
    Criteria
    your IEF gel is cast.

  27. Transfer IEF gel to SDS PAGE gel and run samples.
    Assessment Strategies
    through the completion of laboratory assignments.
    through the completion of written documentation and explanation of laboratory work.
    through the completion of written explanations of laboratory data.
    through completion of an oral report to class.
    Criteria
    you demonstrate completed two-dimensional gel.

  28. Explain purpose and operation of HPLC.
    Assessment Strategies
    through the completion of written assignments.
    through written examinations.
    Criteria
    you give correct explanation according to checklist.

  29. Run samples on HPLC.
    Assessment Strategies
    through the completion of laboratory assignments.
    through the completion of written documentation and explanation of laboratory work.
    through the completion of written explanations of laboratory data.
    through completion of an oral report to class.
    Criteria
    your mw markers are separated and identified.
    you separation of proteins results in clear peaks.

  30. Maintain records of laboratory procedures and array results.
    Assessment Strategies
    through the completion of laboratory assignments.
    through the completion of written documentation and explanation of laboratory work.
    through the completion of written explanations of laboratory data.
    through completion of an oral report to class.
    Criteria
    you maintain records for entire unit according to checklist.

  31. Compile and organize results from purification project.
    Assessment Strategies
    through the completion of written documentation and explanation of laboratory work.
    through the completion of written explanations of laboratory data.
    through completion of an oral report to class.
    Criteria
    all aspects are included.
    your data is organized in tabular form.
    your graphs and gels are present and labeled.
    your calculations are correct.

  32. Present data from purification project.
    Assessment Strategies
    through the completion of written documentation and explanation of laboratory work.
    through the completion of written explanations of laboratory data.
    through completion of an oral report to class.
    Criteria
    your presentation is clearly organized as in 9.2.
    your calculations are shown in stepwise form.

  33. Discuss purification project in a critical fashion.
    Assessment Strategies
    through the completion of written documentation and explanation of laboratory work.
    through the completion of written explanations of laboratory data.
    through completion of an oral report to class.
    Criteria
    you participated with comments during lab presentations in competency 8.2.
    you interpret calculations correctly.
    you interpret graphs and gels correctly.
    you point out inconsistencies and questions in data.

  34. Communicate project results in written report.
    Assessment Strategies
    through the completion of laboratory assignments.
    through the completion of written documentation and explanation of laboratory work.
    through the completion of written explanations of laboratory data.
    through completion of an oral report to class.
    Criteria
    your specific activity is summarized and explained for the entire project. Problems and poor results are discussed. Suggestions for changes in future work given.
    all criteria in 9.2, 9.3 and 9.4 are met in writing.

This Outline is under development.