Biotechnology Laboratory II

COURSE: Biotechnology Laboratory II COURSE NUMBER: BIOT 2933 INSTRUCTOR: Dr. Fabiola Janiak-Spens PHONE: (405) 682-1611, ext. 7414 EMAIL: Fspens@occc.edu ______________________________________________________________ LEARNING MATERIALS: Fundamental Laboratory Approaches for Biochemistry and Biotechnology by Alexander Ninfa and David Ballou. 1998. Fitzgerald Science Press. COURSE DESCRIPTION Prerequisites: (W), (M), BIOT 2823 Corequisite: 3 CREDITS. This continuation of BIOT 2823 will include protein purification. Students will partially purify enzymes, then analyze and assay the resulting purified product. Immunology will be examined using Western blot methods. COURSE COMPETENCIES: Upon completion of this course you will be able to describe and perform a number of steps involved in the purification of proteins such as disruption of cells, ammonium sulfate precipitation, size-exclusion chromatography, ion-exchange chromatography, and affinity chromatography. You will be able to describe and perform Lowry and Bradford assays for determining protein concentrations, colorimetric enzymatic assays, and vertical gel electrophoresis. Finally, you will be able to describe and perform Western blots. COURSE PACING BIOT 2933 is a group-paced course. Your instructors will establish testing and assignment dates. These dates must be observed in order to maintain good standing in the class. COURSE GRADING CRITERIA A: 90-100% average B: 80-89% average C: 70-79% average D: 65-69% average F: Below 65% average Grades will be based on your performances on a combination of classroom tests, presentations, and assignments. SOURCE OF CRITERIA The objectives and competency levels for this course are based upon the professional judgment of Biotechnology faculty and upon comparable courses from other institutions of higher education. ATTENDANCE POLICY: You are expected to attend and actively participate in all scheduled classes for this course. ACCOMMODATIONS FOR STUDENTS WITH SPECIAL NEEDS Oklahoma City Community College complies with Section 504 of the Rehabilitation Act of 1973 and the American with Disabilities Act of 1990. Students with disabilities who need accommodations should make their request in the following ways: · Talk with your instructor after class or during office hours about your disability or special needs related to work in class, AND · Complete the Services to Students with Disabilities Intake/Referral Form provided in the Office of Services to Students With Disabilities (unless this has been completed). · For additional information, call 682-7250 V/TTY COURSE OBJECTIVES Proteins 1. The student can recognize the different amino acids and classify as aliphatic, aromatic, basic, acidic, sulfur-containing, or hydroxyl-containing, and knows the three-letter and one-letter abbreviations for the amino acids. 2. The student can show how amino acids react to form peptide bonds. 3. The student can describe the four levels of protein structure and the different forces involved in maintaining protein structure. The student can describe what happens when proteins are denatured. 4. The student is able to describe the methods used for detecting proteins (including A280's) and determining protein concentrations, and discuss the differences between Biuret, Lowry, and Bradford assays. 5. The student performs Lowry and Bradford protein assays, using a spectrophotometer, and graphs a standard curve using Excel. Student can determine the concentration of an unknown from the standard curve. 6. The student is able to describe the basis of ion-exchange chromatography and distinguish between cation and anion exchange chromatography. The student can prepare a protein extract and make the necessary buffers for running an ion-exchange column. The student can describe and perform the different stages of running an ion-exchange column: preparing the resin, pouring the column, washing the column, loading the column, changing buffers, and collecting fractions with aid of fraction collector. The student uses Microsoft Excel to plot the absorbance at 280nm of the fractions or the enzymatic activity of the fractions versus fraction number or elution volume. 8. The student can describe the basis of polyacrylamide gel electrophoresis (PAGE). The student is able to describe the differences between native gels and denaturing gels, between non-reducing gels and reducing gels, between gradient gels and straight gels, and between stacking gels and resolving gels. The student can interpret the banding patterns from any of the listed gel types, or combinations of gel types. The student knows the hazards associated with PAGE and why the different ingredients are needed in the gel recipes and in sample preparations. 9. The student learns to cast a polyacrylamide gel. 10. The student will be able to describe ammonium sulfate precipitations. Given a table, the student will be able to calculate the amount of solid ammonium sulfate needed to raise a given volume of sample to a certain percentage of ammonium sulfate saturation. The student will be able to use centrifugation to separate precipitated proteins from the supernatant. 11. The student will be able to describe the basis of gel filtration (size exclusion chromatography). As with ion exchange chromatography, the student will be able to describe the different stages of running a gel filtration column. In addition, the student will be able to describe what is meant by excluded volume, included volume, and bed volume. The student will be able to use Microsoft Excel to plot the absorbance at 280 nm of the fractions or the enzymatic activity of the fractions versus fraction number of elution volume. Given proteins of known molecular weights, the student will be able to use gel filtration to determine the molecular weight of an unknown protein. 12. The student will be able to describe affinity chromatography and describe the different stages of running an affinity column and plot absorbance against fraction volume or number using Microsoft Excel. 13. The student will be able to conduct a colorimetric enzyme assay to determine the enzyme's relative activity per mL. After being given protein concentration or determining protein concentrations from standard curves, the student will be able to calculate the specific activity of an enzyme. 14. The student will be able to outline a multi-step protein purification, describing where the desired protein is at any given point in the process. The student will be able to calculate the following for enzyme samples at different stages of a multi-step purification: protein concentration, total protein, activity per mL, total activity, specific activity, percent recovery, fold-purification. 15. The student will be able to do enzyme assays in the presence and absence of an inhibitor using different substrate and inhibitor concentrations. The student will be able to use Microsoft Excel to generate Lineweaver-Burke plots to determine Vmax and Km values for an enzyme. Oklahoma City Community College Biotechnology Program Dr. Fabiola Janiak-Spens, Program Director 7777 S. May Avenue Oklahoma City, OK 73159-4444 (405) 682-1611, ext 7414 Comments: Fspens@occc.edu Copyright 1998 Oklahoma City Community College 1/06