CHEM 420 BIOSEPARATIONS

Individuals entering a career in biotechnology will frequently need to be able to separate biological molecules, other cellular and tissue components, and products from genetic engineering protocols from a more complex sample.  Such separation techniques are needed for sample analysis, preparation and synthesis, and purification in both industrial applications and research.

I.                    Course description:

     This laboratory-intensive course emphasizes key analytical and preparative separation techniques used in biochemistry and biotechnology.  Theoretical, practical and instrumental components of the methods will all be emphasized.

     As an advanced laboratory course, students will be expected to read and extract protocols from primary journal articles.  Laboratory reports will be prepared in journal manuscript format and some results will be presented in poster format for class discussions.

     Teams of students will be asked to perform a isolation and separation and characterization on of 3 of the 4 broad classes of biomolecules: lipids, proteins carbohydrates and DNA.    Projects may pass from team to team if appropriate.  

     Teams will conference once a week with the professor to report progress and help plan for future work. Set up meeting times first week of each project. 

     Teams may work outside specified Laboratory hours on the following conditions:

 First no other class should be using Scitech 360. 

 Second If they are using equipment from other laboratories they must have the permission of any instructor using the laboratory at that time. 

 3.  Students must sign in and out for a minimum of six hours of laboratory a week.  Sign in sheets will be posted in the laboratory.  Students should sign in and out during regularly schedule laboratory hours so that they are counted for the week.   Weekly Hours Sheets  (Due Mondays for previous week.)  

Posted Quizes  Buffers 1 Key,   Buffers 2, Buffers 2 KEY Buffers 3Quiz  Quiz 3 Key  Quiz 4  Quiz 5 Quiz 6

II.                 Course objectives:

Methods development

Converting information from a Paper to a Protocol.

Keeping a laboratory note book

Learning practical buffer and reagent preparation.  

Learning methods of biochemical separation.

III.               Course Requirements:

Ø      attendance and participation

Ø      Mantain a detailed Laboratory Notebook 

Ø   Prepare for and conduct experiments

Ø      follow all laboratory safety protocols

Ø      preparation of laboratory reports and/or poster presentations

Ø      journal article readings and critiques

Ø      quizzes:  There will be at least one quiz on the syllabus additional quizzes will be given as warranted. 

Ø      Adherence to the code of academic conduct:  Please follow the link on the main page and print and sign the pledge. Infractions of the code will result in penalties up to and including failure of the course

IV.              Course Textbook:

You will need a Bound Laboratory manual.  Bound this means it leaves a permanent record if something is removed More on this item later.  You should plan on copying/printing about 500 pages of material during the course of the semester.  I would recommend a three ring binder.  3 inches should do you.   

This is a course in methods development.  Students will be asked to research and develop methods to effect a separation on mixtures identified by the instructor.   The projects and papers below are starting points .    The endpoint  is what your grade depends on.   there are two reqired projects and two optional projects.  Each project  will take about  4 weeks to complete. 

Projects   Lab  book one  report

1.  Lipids 400 pts (required)

            Isolation and separation of Terpenes from juniper, white pine, or eastern red cedar. Zhou J. and Cates R. G. 1995.  Journal of Chemical Ecology  21:387

      Dertermination of adulteration of Olive oils  Blanch et al 1998J. Agric. food Chem 46: 3153-3157

Determination of lipid composition of Olive Oils  Ollivier D 2003 J. Agric. Food chem.51:5723-5731

2. Proteins 400 pts (required)

      Isolation of and characterization of ferridoxin, BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION

 Vol. 31, No. 2, pp. 119–122, 2003  (This procedure does not yeild pure protein you should spend some time determining how to do so.)

3.  Carbohydrates 200 pts

Isolation and characterization of Glycogen from multiple sources (Calf and chicken liver you will be doing end group analysis) Clark Experimental biochemistry 1978

4.  DNA 200 pts

      Isolation and characterization of a plasmid. 

Bibliography:

Books:

The following would prove to be helpful and could be put on reserve in the library or my office during the semester that the course is offered at students request:

Protein Purification Principles and Practice 3^rd Ed.  (1994)  Scopes Robert K.  Springer Verlag, Berlin

Methods of Protein and Nucleic Acid Research: Chromatography.  Lev A. Osterman Springer Verlag, Berlin

Fundamental Laboratory Approaches for Biochemistry and Biotechnology, A.J. Ninfa and D.P. Ballou, Fitzgerald Science Press, Inc. (Bethesda) 1998.

Basic Methods in Molecular Biology, L.G. Davis, M.D. Dibner, and J.F. Bailey, Elsevier Press (New York) 1986.

*Handbook of biochemistry and molecular biology, Gerald D. Fasman, ed.

CRC Press, (Cleveand)  1975 - 1977.

*Applications of high performance liquid chromatography, A. Pryde and M. T. Gilbert.

Halsted Press, 1979.

*Practical high performance liquid chromatography, edited by C. F. Simpson,

Continuing Education Committee of the Chemical Society, (London) 1976.

*Separations by centrifugal phenomena, Hsien-Wen Hsu ; editor, Edmond S.

Wiley Publishers (New York), 1981.

*Foundations of ultracentrifugal analysis, Fujita, Hiroshi, Wiley Publishers (New York), 1975.

NOTE:  Due to the rapidly changing technology in this field, the best resources for this course will be current primary literature.  ESU has a significant number of periodicals containing relevant material in protein chemistry.

Periodicals:

Biotechniques

*Journal of chromatography, Elsevier Press, 1958 -

*Chromatographic reviews, Elsevier Pres 1959 -

*Methods of Enzymology, Academic Press (San Diego, New York) 1955 -

*Annual review of biochemistry, 1932 -

*Biochemical journal, 1906 -

*Biochemistry, 1962 -

*Journal of biological chemistry, 1905 -

*Journal of molecular biology. 1959 -

*Methods of biochemical analysis, 1954 -

Other Resources:

Protein Protocols on CD-ROM (Humana Press)

            Resource obtained from ESU Foundation Grant

            Installed on student computers in 360 scitech

Interactive Biochemistry CD-ROM (Saunders College Publishing)

            Installed on student computer in 360 scitech

Course Content

Topics to be discussed and or literature to be reviewed:  Lectures once weekly on Monday's  Content/Schedule may change as needed.

A.        Differential Solubility

            1.         Extractions

                        Steam distillations.

            2.         Precipitations:  effects of salts (ionic strength), pH and solvent polarity

            3.         Crystallization. 

B.         Chromatography Basics

            1.         Theory and principles

            a)         Partitioning and adsorption

b)         Theoretical plates

c)         An overview of the types of chromatography

            2.         Hardware

                        a)         Columns

b)         Inert supports/matrices

c)         Resins

d)         Detectors

3.         Column Chromatography Methods

a)         Ion exchange

b)         Gel filtration/molecular sieving

c)         Affinity chromatography

                        d)         Reverse phase methods

                        e)         Hydrophobic interaction chromatography

                        f)          Chiral separations

4.         Medium and High Pressure Liquid Chromatography

C.  Applications of Chromatography in Biochemistry and Biotechnology

            1.         Coupling chemistry/Resin development

            2.         Sample preparation

            3.         Sample monitoring

4.         Protocols for Proteins, Nucleic Acids, Carbohydrates, Lipids, Organic acids

            5.         Biochemical applications of GC-MS

D. Dialysis and Filtration

            1.         Theory

            2.         Dialysis tubing/cassettes

            3.         Filters and membranes

            4.         Ultrafiltration

            5.         Microconcentrators

E. Centrifugation  (covered in Biophysical chem)

1.                  Theoretical aspects:

2.                  Buoyancy, gravitation and centrifugal forces

3.                  Sedimentation vs. equilibrium

4.                  Differential centrifugation

5.                  Density gradient centrifugation

6.                  Determination of molecular weight, sedimentation coefficients and frictional coefficients

F. Electrophoresis

1.                  Theory

2.                  Methods

a)                  Discontinuous vs. continuous

b)                  Vertical and horizontal methods

c)                  Isoelectric focusing

d)                  Native and Denaturing methods

e)                  Capillary electrophoresis

            3.         Applications

G. Cell and Organelle Separation Techniques

1.                  Differential centrifugation

2.                  Flow cytometry and cell sorting

Reagents: 

            You will be responsible for identifying the reagents/buffers needed for your work.  You may place orders for reagents to the stockroom through the instructor, or prepare them your self.  Some reagents may not be in stock, please check the inventory lists in the laboratory.  Reagents must be requested at least 1 week in advance of there use. 

Grades: Grades will be assigned at the pleasure of the instructor.  The following point percentages will result in the corresponding minimum grades  because they presumably demonstrate the corresponding understanding.

Comments on Notebook Keeping and the lab:

  I am hitting some highlights here; a more complete treatise on notebook keeping is linked here.  Read both; what is covered here is C-B level of notebook keeping and is a minimum to pass the course.  

            A laboratory notebook MUST be kept. This notebook should have a permanent binding with numbered pages (if not already numbered, you may do so in ink).  You do not need duplicate tear out pages for this course.  Three ring binders or spiral notebooks are not acceptable. If you are unsure whether a book is appropriate, show it to me BEFORE using it.

            A table of contents MUST be included; keep the table of contents current. Use pen only to write in your notebook.

            Never enter anything in pencil. Never erase or scribble out any entry your notebook.  Use of white out in a notebook will result in 0 credit for the days work.  If an error is made cross it out using a SINGLE line through the error, INITIAL and DATE the cross-out and write the correction above, below or to the right of the error. The error MUST be readable even after correction.

            You must use indelible ink to write in the notebook.  Use of a felt tipped marker is not acceptable. You laboratory notebook will probably get wet at least once this semester. Marker ink runs and smears when wet.

            NEVER enter your laboratory data on a separate sheet for later entry in the notebook. Data MUST be entered in the notebook directly. Print outs should be taped into the notebook with the initials date and page written on the print out. The print out should be initialed and dated such that it spans both the print out and the page it is taped to. If you are working in pairs you should refer to your partner’s notebook and include a copy of the data in your notebook treated in the same fashion as the original data.  Notebooks will be collected during the semester and checked for completeness of preparatory work and collected again after laboratory on a irregular basis to check on completeness of data collection. The grade will be based on organization and completeness.  Notebooks should be readable; however, as this is a working document, I realize that it will not be as neat as a handed in assignment. 

            All calculations which are part of the laboratory book MUST be included in your notebook. Incomplete notebooks will be penalized. Do not leave the laboratory without a complete record of your data. Do not rely on a laboratory partner to keep the data or provide data later. Lack of data or problems with a lab partner are not valid excuses for late or incomplete assignments.

Preparation for Lab:

YOU MUST COME TO LAB PREPARED. This includes reading, studying, planning and preparing the laboratory in your notebook.  Your notebook must contain your preliminary procedure and instructions/calculations needed to complete the work.           All calculations, which are part of the laboratory, MUST be included in your notebook.  This procedure should be included in your laboratory notebook in the format suggested.  The procedure should be in your own words, not copied directly from the text/web or other handouts/papers.  You may bring your handouts and text to lab for reference; however, you should follow the procedure as directed in your notebook

There should be prepared space for recording data.  This often may take the form of tables.  Leave room for print outs You will probably want to use whole pages for these. 

Completing these labs in the time required will be challenging. If you are unprepared, it will be impossible.  If you are unprepared you will be excused from lab for that day and a grade of 0 will be recorded for that laboratory, no exceptions. Laboratory notebooks will be worth 50% of each laboratory.

Grading of laboratory notebooks: 1 hour or more prior to work the notebook should be dropped of at my office for review if you are working on Monday  or Wednesday you should drop it off before noon two hours before as it is expected that those days will have heavy loads.  Prior to starting your work I will review the note book and give a percent grade to the notebook.  If I have the time I will write notes in the note book to help you along.  You  must receive a 70% or better to proceed.  At the end of a project you will receive a point total based upon the percent grades in your note book. 

Laboratory Reports and Due Dates:

Each laboratory report will be worth 50 % of the points for a given assignment. Laboratory reports must be typed/word processed. They WILL NOT be accepted hand written. Laboratory report will be due on two weeks after the last laboratory experience on the project.