• BIOC 3570 Analytical Biochemistry Fall 2005


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    • Abstract: There is little or no coverage of physical methods such as spectroscopy, centrifugation, ... QP519.7 .R63 Good coverage of chemical-analytical techniques for specific. classes of biomolecules, including topics we won\'t discuss, such as lipid ...

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BIOC 3570
Analytical Biochemistry
Fall 2005
Instructor: Dr. H. McDiarmid
Office: SC 1241
[email protected]
Website: All course material will be posted on WebCT
Office Hours: TBA
I welcome questions and comments sent by email. I will try to
reply within one working day.
NOTE: If you wish to see Dr. McDiarmid at another
time, please make arrangements via email.
Lectures: Monday, Wednesday 7:00-8:20 pm
ANNU 156
Laboratories:Mon-Fri 1:30-5:30 pm SC 3101
Attendance for the full time of each laboatory is compulsory.
Laboratory sessions start the week of Sept 19 with Experiment 1.
Please be prepared. You must buy a lab manual at least one day
prior to the lab so that you can do the advance preparations. You
will also require goggles, a three-ring binder and a laboratory
notebook. Please see your TA, or Paula Russell (SC 3115, x 58220)
if you have questions concerning the laboratory.
Grading: Midterm exams (2) 30 %
Laboratory 35 %
Final exam 35 %
100 %
Only valid excuses on medical or compassionate grounds will
prevent a grade of zero for any missed labs or exams (see Calendar
Section VIII). Documentation may be required.
Students must pass both the laboratory component and the theory
component to obtain a final passing grade for the course.
Examinations: Midterm Dates Wednesday October 19 in class
Monday Novembe 14 in class
Details of midterm to be announced.
Midterm papers may be returned to me for correction of grading
errors within one week of the return of the paper to the student. I
may refuse to re-grade a paper at my discretion.
The final exam is scheduled for Friday Dec 9, 8:30- 10:30 am.
The final exam will cover material from the entire course with an
emphasis on the material covered after the second midterm.
Prerequisites: [CHEM 2400 or CHEM 2480] and [CHEM 2580]
Familiarity with the following basic aspects of biochemistry is
assumed : protein and nucleic acid structure, including the
structures of amino acids; flow of genetic information; basic
enzymology. Please review this material carefully, especially if
some time has passed since you took CHEM 2580.
If you have not taken both genetics courses MBG 2000 and MBG
2020, you should carefully read the chapter on Recombinant DNA
Technology in Lehninger 4th edition (or corresponding chapters in
another text), as soon as possible. Several sections of the course
require familiarity with concepts from physics. We will review
these in class, but independent study may also be helpful.
Method of
Presentation: In addition to lectures, there will be occasional tutorial/problem
sessions. Problem assignments will be handed out in class and
posted on the web. To obtain benefit from these exercises, it is
essential that you attempt the problems on your own before
attending the tutuorial session. Solutions will be posted on the
website after the tutorial session.
Objectives: This course provides theoretical and practical knowledge about
commonly used laboratory techniques used in biochemistry.
Methods relating to the expression, isolation, separation and
analysis of proteins are the focus of this course, although
applications for other biomolecules are included.
Adhering to the University of Guelph Learning Objectives
(Calendar, pp. 2-4), the course (and especially its laboratory
component) will help to develop the student’s numeracy (obj. 2),
understanding of forms of inquiry (obj. 7); and independence of
thought (obj. 9). The writing of laboratory reports will strengthen
the component of literacy (obj. 1). We will also consider the
historical development of the science (obj. 3) and the relationship
of biochemistry to other physical and biological sciences (obj. 8).
Readings There will be one journal article assigned for reading during the
semester. This reading is mandatory and questions relating to it
may appear on exams.
Texts:
We have tried various textbooks in previous offerings, but none has proven
satisfactory; either the level of coverage or the selection of topics is a poor fit. We will
not be using a required text. However, absent a textbook, there is additional onus on you
to study independently and to seek out other sources of information. A list of relevant
books (some of which have previously been used as texts) is given below.
You will need to consult a comprehensive biochemistry textbook; the latest
editions of the texts by Lehninger, Nelson, and Cox (3rd ed., 2000), Stryer (5th ed., 2002),
or Zubay et al. (4th ed., 1998) are recommended. We also cover several topics which are
based on concepts from physics, so you will need to consult a physics textbook from time
to time.
The following books are recommended. The ones marked * *are available at the
library reserve desk; those marked * are available in the stacks. Please see Dr.
McDiarmid if you require further references.
* A.J. Ninfa and D.P. Ballou, Fundamental Laboratory Approaches for Biochemistry and
Biotechnology, Fitzgerald Science Press, Bethesda, MD, 1998. This book is up-to-date
and well-written. The sections on protein purification techniques are the strongest.
There is little or no coverage of physical methods such as spectroscopy, centrifugation,
and radioactivity.
** D.J. Holme and H. Peck, Analytical Biochemistry (3rd ed., 1998) Longman
(QP 519.7.H64). This book has some good practical information on laboratory
techniques, but the theoretical coverage is weak.
** D. Freifelder, Physical Biochemistry (2nd ed., 1982) Freeman (QH 345.F72) This
book is an excellent reference text, especially with respect to the theory of spectroscopy,
centrifugation, electrophoresis, and other biophysical techniques. Freifelder covers in
detail specialized topics that are rarely found in other books, such as determination of
partial molar volumes of proteins. But the book is too advanced for routine
undergraduate use and it is now badly out-of-date.
** R.F. Boyer, Modern Experimental Biochemistry (2nd ed., 1993) Benjamin
(QP519.7.B68)
This is a rather low-level textbook. The second half consists of laboratory exercises.
** M.P. Deutscher, ed., Guide to Protein Purification, Methods in Enzymology vol. 182,
Academic Press, 1990. An authoritative collection of review articles on all aspects of the
title subject.
** J.F. Robyt and B.J. White, Biochemical Techniques : Theory and Practice, Waveland
Press, 1990. QP519.7 .R63 Good coverage of chemical-analytical techniques for specific
classes of biomolecules, including topics we won’t discuss, such as lipid analysis.
REFERENCES FOR BIOCH 3570 lab. -- available at library reserve desk:
Bradford, M., A rapid and sensitive method for quantitation of microgram quantities of
protein utilizing the principle of protein-dye binding. (1976) Anal. Biochem. 72:248-254
Castellino, F.J. and Hill, R.J., The carboxymethylation of bovine -lactalbumin (1970) J.
Biol. Chem. 245:417-424
Compton,S. and Jones, C. G., Mechanism of dye response and interference in the
Bradford protein assay (1985) Anal. Biochem. 151:369-374
Dallas, M.S.J, Reproducible Rf values in thin-layer adsorption chromatography (1965) J.
Chromatog. 17:267-277
Ebner, K.E., A biological role for -lactalbumin as a component of an enzyme requiring
two proteins (1970) Acct. Chem. Res. 3:42-47
Edelhoch, H., Spectroscopic determination of tryptophan and tyrosine in proteins (1967)
Biochemistry 6:1948-1954
Herrington, C.S., et. al., Interphase cytogenesis using biotin and digoxigenin labelled
probes I: Relative sensitivity of both reporter molecules for detection of HPV16 in CaSki
cells (1989) J. Clin. Pathol. 42:592-600
Kronman, M.J. and Andreotti, R., Inter- and intramolecular interactions of -lactalbumin.
Part I. The apparent heterogeneity at acid pH (1964) Biochemistry 8:1145-1151
Kronman, M.J. and Andreotti, R., Inter- and intramolecular interactions of -lactalbumin.
Part II. Aggregation reactions at acid pH (1964) Biochemistry 8:1152-1160
Laskey, R.A. and Mills, A.D., Quantitative film detection of 3H and C in
14
polyacrylamide gels by fluorography (1975) Eur. J. Biochem. 56:335-341
Lui, T.Y. and Chang, Y.H., Hydrolysis of proteins with p-toluenesulfonic acid (1971) J.
Biol. Chem. 246:2842-2848
Matthews, J.A. and Kricka, L.J., Analytical strategies for the use of DNA probes (1988)
Anal. Biochem. 169:1-23
Mullis, K.B., The unusual origin of the polymerase chain reaction (1990) Scientific
American 262:56-65
Phillips, N.J. and Jenness, R., Isolation and properties of human -lactalbumin (1971)
Biochim. Biophys. Acta 229: 407-410
Weil, L. and Siebles, T.S., On the structure of -lactalbumin: Degradation studies with
carboxypeptidase A and carboxypeptidase B (1961) Arch. Biochem. Biophys. 93:193-200
Wong, E., et. al., Cloning of a turkey prolactin cDNA: Expression of prolactin mRNA
throughout the reproductive cycle of domestic turkey (Meleagris gallopavo) (1991) Gen.
Comp. Endocrin. 83:18-26
Yang, V.C. and Langer, R., A simple and economical technique for Pi measurement
(1987) Biotechniques 5:138-143
OTHER REFERENCES (mainly, manuals from commercial suppliers)
Affinity Chromatography; Principles and Methods; Pharmacia Biotech 1988
Biospec Number 1: “An introduction to protein electrospray mass spectrometry”, M.J.
Geisow and B.N. Green.
Biospec Number 3: “Protein mixture profiling: Applications of an improved method of
presenting electrospray data from protein mixtures”, J.C. Cottrell and B.N. Green
Dig System User’s Guide for Filter Hybridization; Boehringer Mannheim (Roche) 1995
Gel Filtration: Theory and Practice; Pharmacia Biotech 1989
GST Gene Fusion System; Pharmacia Biotech 1993
Nucleic Acid Isolation and Purification; Boehringer Mannheim (Roche) 1998
Protein Electrophoresis, Technical Manual; Amersham Pharmacia Biotech
Sequencing Chemistry; Amersham Pharmacia Biotech 1999
Topics covered
PART A Protein Expression
1. choice of organism
2. transfections/transformations
3. overexpression of proteins
PART B Protein Purification
1. Theory
2. Purification methods
PART C UV-Vis Spectroscopy and Fluorescence
1. Theory
2. Instrumentation
PART D Electrophoresis
1. Theory
2. Applications
3. Staining of protein gels
4. Band detection on nucleic acid gels
PART E Protein sequencing
1. Mass spectroscopy
2. Whole protein sequencing
PART F Immunotechniques
1. Theory
2 Applications
PART G Use of Radioisotopes
1. Theory
2. Instrumentation
PART H Centrifugation
1. Theory
2. Differential centrifugation
3. Rate-zonal sedimentation
4. Equilibrium sedimentation


Use: 0.0892