King Saud University
Department of Biochemistry
Techniques in Biochemistry course
(BCH 530)
Protein Purification technique
1st Semester 1431/2010
Course Outline
Instructor(s):
Dr. Sooad Aldaihan
Dr. Samina Hyder Haq
Dr. Abeer Alghanouchi
Office no. 704 e-mail: sdaihan@ksu.edu.sa
Office no. 702 e-mail: shaq@ksu.edu.sa
Office no. 674 e-mail: aalghanouchi@ksu.edu.sa
Course objectives:
This open-ended laboratory based course covers a wide variety of conventional
methods for protein isolation, purification and characterization. Different techniques of
protein separation will be discussed and practiced in the lab in an integrated manner. The
objective of this is to explain and emphasize how strategies could be developed for
protein isolation and purification. Students will use various protein purification Protocol
and characterization techniques including chromatographic and electrophoretic
techniques. A special feature of the course is that all laboratory work will be performed
individually where the efficiency of sequential purification steps will be evaluated by
constructing and analyzing purification tables. Protein and enzyme assays will be studied
and used during the purification process. The basic principles of enzyme kinetics and
determining kinetic parameters using different methods will also be covered in this
course. Alpha amylase from yeast and Acid Phosphatase from an animal tissue will be
used for Enzyme purification and characterization.
Evaluation & Grading:
The evaluation is based on the:
a) Reports (30%).
b) Final exam on the theory and practicum (50% that include oral exam and
presentation of their work in the form of a poster)
c) Attendance, participation, accuracy and independence during the practicum (20%).
Text Book:
Protein Purification Methods: a practical approach (Harris & Angal, IRL Press), 1989.
References:
1. Hatti-Kaul, R., and Mattiasson, B. (2004). Isolation and Purification of Proteins.
Marcel Dekker Inc., New York, NY.
nd
2. Walker, John M. (2002). The Protein Protocols Handbook, 2 edition. Humama
Press, Inc., Totowa, NJ.
3. Doonan, Shawn, ed. (1996) Protein Purification and Protocols. Methods in
Molecular Biology, 59, Humana Press, Inc., Totowa, NJ.
4. Janson, J-C., and Rydén, L. (1989). Protein Purification. Principles, High
Resolution Methods, and Application. VCH Publishers, Inc.
5. Jacoby, William B. (ed.) (1984). Enzyme Purification and Related Techniques.
Methods Enzymol., Vol. 104, Academic Press, San Diego, California.
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6. Scopes, Robert K. (1994). Protein Purification, Principles and Practice, 3nd Ed.
Springer-Verlag, New York.
7. Ahmad, Hafiz (2005). Principles and Reactions of Protein Extraction, Purification,
and Characterization. CRC Press (Taylor & Francis).
Outline:

Preparation of crude extract

Protein assay

Lowry method.

Bradford method.

UV absorption using A280nm & A260nm.

ACP activity assay using para-nitro phenyl phosphate as a substrate.

Amylase activity assay using starch as substrate.

Enzyme kinetics and determining the kinetic parameters (Km & Vmax) for ACP and
amylase using different plots.

Purification using ammonium sulfate precipitation (salting out).

Desalting methods (dialysis and gel filtration).

Purification using column chromatography.

Gel-filtration (Size-exclusion) column chromatography.

Ion-exchange column chromatography (Sephacryl MonoQ)

Constructing and analyzing purification tables.

SDS–Polyacrylamide gel electrophoresis (SDS-PAGE).
Work Strategy:
The Students will be divided into 3 groups as following:
Group
Instructor
Experiment
Location
Gp 1 (~5 students)
Dr. Sooad
Amylase purification
Building 20
Gp 2 (~5 students)
Dr. Samina
ACP purification
Lab. No.7
Gp 3 (~5 students)
Dr. Abeer
Chromatography and
electrophoresis techniques
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