The Biotechnology Curriculum Collection of the California Community Colleges

ORANGE COAST COLLEGE COURSE OUTLINE OF RECORD

Course developer: Farah Sogo
Course static ID:
TOP No. 1905.00
CIP No.
Course adoption:
Date revised:
Revised by:
Semester(s) offered: Spring
Distance education alternative

COURSE MASTER DICTIONARY DATA

Title 5 credit status: Associate degree credit course X
Nondegree credit course
Noncredit course
Course name/number: Biology 281L/Chemistry 281L
Division: Math and Science
Course title: Protein Chemistry Laboratory
Department: Biology or Chemistry
Units: 1
Total course hours: 54
Course length: 1 semester
Weekly hours configuration: 3 hours laboratory per week
Grading method: Graded X
CR/NC
Student option
Noncredit
Method of Instruction: 20 (2 digit no.)
Basic skills status: N (P, B or N)
Materials fee: No X
Yes $ 4.00

Justification: Gels and nitrocellulose membranes are used to analyze protein, and for permanent reference and technique records.

COURSE PREREQUISITE:
Biology 281 or concurrent enrollment.

CATALOG DESCRIPTION:
This laboratory course will focus on experimental methods involving the purification and analysis of protein. The students will gain hands on experience with spectophotometry; enzyme purification and kinetics; ion exchange, affinity, and gel filtration chromatography; SDS-Polyacrylamide electrophoresis; and Western blot analysis.

SCHEDULE DESCRIPTION:
This laboratory course will focus on experimental methods involving the purification and analysis of protein.

COURSE CLASSIFICATION:
A Liberal arts/AA X
B Remedial
C Remedial
D-H Community course
I Occupational required
I Occupational elective

COURSE TRANSFER:

0 Non-transfer/Non-AA
1 Non-transfer AA
2 Transfer CSU X
3 Transfer UC

JUSTIFICATION FOR THE COURSE:
Required for certification
Occupational State of the art.
Advisory committee recommendation

COURSE CONTENT AND SCOPE/TOPIC OUTLINE:

1) Acids, Bases and buffers

a) Topics covered

i) Strong acids and bases
ii) Weak acids and bases
iii) Polyprotic acids
iv) Buffers
v) Good's buffers
vi) Choosing a buffer
vii) Effect of concentration and temperature
viii) Making buffers

b) Experiment

i) Preparation of buffers

2) Spectrophotometry

a) Topics covered

i) Absorption of Light
ii) Beer-Lambert Law
iii) Standard Curves
iv) Protein Assays
v) Calculating Concentrations from graphs

b) Experiments

i) Beer's Law and Standard Curves
ii) Protein Concentration of Lactate Dehydrogenase (LDH) Fractions

3) Enzyme Purification

a) Topics covered

i) Enzymes as Catalysts
ii) Enzyme Purification
iii) Units of Enzyme Activity
iv) Calculating Initial velocity
v) Purification tables
vi) Assay LDH

b) Experiments

i) Purification of LDH

4) Ion Exchange Chromatography

a) Topic covered

i) Amino acids as weak acids and bases
ii) Isoelectric point
iii) Ion exchange chromatography
iv) Ion exchange resins
v) Identification of compounds eluted from columns
vi) Thin layer chromatography

b) Experiments

i) Separation and identification of amino acids
ii) Purification of LDH with ion exchange chromatography

5) Affinity Chromatography

a) Topics covered

i) Affinity chromatography
ii) Gel supports
iii) Affinity ligands
iv) Elution of bound molecules

b) Experiment

i) Affinity chromatography of LDH

6) Gel Filtration chromatography

a) Topics covered

i) Gel filtration
ii) Types of supports
iii) Determining the molecular weight
iv) Distribution coefficients

b) Experiments

i) Gel filtration chromatography
ii) Gel filtration chromatography of LDH

7) Enzyme Kinetics

a) Topics covered

i) Reaction rates
ii) Order of reaction
iii) Michaelis-Menten Approach
iv) Significance of Km and Vmax
v) linear plots
vi) properties of tyrosinase

b) Experiments

i) Enzyme kinetics of tyrosinase
ii) Enzyme kinetics of LDH

8) Western Blots

a) Topics covered

i) Theory
ii) Antibodies
iii) Color development
iv) Blocking and washing

b) Experiments

i) Western blots of serum proteins
ii) Western blot of LDH

9) Electrophoresis

a) Topics covered

i) Electrophoresis
ii) Agarose Gels
iii) Polyacrylamide Gels
iv) SDS-PAGE
v) Staining Gels
vi) Lactate Dehydrogenase

b) Experiments

i) Native Gel separation of LDH isozymes
ii) SDS-polyacrylamide gel electrophoresis

INSTRUCTIONAL OBJECTIVES:

The students will be able to:

1) Properly make, use, and choose buffers.
2) Use a spectrophotometer to produce standard curves and determine protein concentration from them.
3) Calculate absorbencies, concentration and extinction coefficients using Beer's law..
4) Purify the enzyme lactate dehydrogenase (LDH) from beef heart and measure enzyme activity.
5) Determine the specific activity of enzyme fractions and construct a purification table for the purification of LDH.
6) Explain how anion and cation exchange columns work and predict the order of elution of amino acids off an ion exchange column.
7) Apply samples properly to columns and thin layer chromatograms.
8) Prepare a Cibacron Blue Sepharose column and assay a sample for LDH, calculate relative activity and calculate the number of units loaded on the column.
9) Separate amino acids by cation exchange chromatography and identify them by thin layer chromatography (TLC).
10) Properly load a sample on a Sephadex gel without disturbing the gel bed and measure the peak elution volumes of given samples.
11) Observe and analyze the effect of changing enzyme concentration and changing substrate concentration on initial reaction rates
12) Design protocol for determining kinetic parameters.
13) Pour and load an agarose gel for electrophoresis
14) Identify LDH isozymes based on their electrophoretic migrations
15) Pour polyacrylamide gels and assemble the gel chamber, Prepare and load protein samples in the sample wells, and stain and destain the gels with Coomassie Blue.
16) Set up a Western blot apparatus and gel sandwich, and react blots with primary and secondary antibodies.

METHOD OF STUDENT EVALUATION:
Student written exams, completion of laboratory reports, completion of problem sets.

INSTRUCTIONAL METHODOLOGIES:
Prelaboratory lectures on key concepts.
Hands-on laboratory experiments
Weekly problem sets.

WRITING ASSIGNMENTS/PROFICIENCY DEMONSTRATION:

The students are required to prepare written laboratory reports and problem sets on a weekly basis.

REPEATABILITY:
N/A

FEASIBILITY:

Faculty: This course may be taught using existing biology or chemistry faculty, or can be taught using adjunct
faculty.
Classroom: This course will be taught in existing classroom facilities.
Library Learning Resources: The library has an interlibrary loan program and Internet access that will be
sufficient for any necessary classroom research.

EDUCATIONAL MATERIALS:

Experiments in Biochemistry: A Hands-on Approach, Farrell, S.O., and Ranallo, R.T, Harcourt Brace and Company.