CHEM 4242: TECHNIQUES IN BIOCHEMISTRY

SPRING 2009; Thursday, 1:00-5:00 pm

Instructor Dr. Subhrangsu S. Mandal

Office CPB 349
Office hour 11 AM - 12 PM
Phone 817-272-3804
E-mail smandal@uta.edu

Teaching Assistant Sahba Kasiri

Office CRB 310
Office hour 10 AM - 12 PM
Phone 817-272-5436
E-mail sahba.kasiri@mavs.uta.edu

Lecture: 125 Science Hall; Time: 1:00 - 1:50 P.M.

Laboratory: 210 CPB; Time: 2:00 - 5:00 P.M.

TEXT BOOK: “Modern Experimental Biochemistry” Third Edition by Rodney Boyer as

well as the handouts

COURSE OVERVIEW AND OBJECTIVES: Welcome to biochemistry laboratory! This

practical course is intended to give you an opportunity to learn the basic techniques
routinely used in biochemistry and molecular biology. The students will be introduced to
the theory that serves the basis for these techniques. It will include discussion of the
theoretical principles, practical details and applications of the key experimental
techniques, including those that have led to the emergence of the new disciplines of
genomics, proteomics and bioinformatics. The students should be able to perform the
biochemical experiments independently and interpret the lab results. Each experiment
will cover the principles of experimental design and the statistical analysis of quantitative
analytical data. To enhance student understanding of each topic, in-text worked
examples are included in each lab. The textbook “Modern Experimental Biochemistry” by
Rodney Boyer as well as the handouts will be given for each lab one week prior and will
be discussed during the lecture hour and lab. Practical applications of all commonly
used analytical techniques will be discussed.
By the end of the course you should be familiar with a number of common
biochemical techniques: use of pipettes, balances and the spectrophotometer; making
buffers, methods of protein determination; analysis of enzyme kinetics; methods of
protein purification; electrophoresis (SDS-PAGE and Agarose gel), plasmid purification,
restriction analysis and polymerase chain reaction (PCR).

STRUCTURE: The lab will be emphasizing the qualitative as well as the quantitative
aspects of the biochemical experiments. This will include: (a) common biochemical
techniques, SDS-PAGE and agarose gel electrophoresis; (b) properties of
macromolecules; and (c) recombinant DNA technology.

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LAB SCHEDULE: For experiments, assignments and due dates, please see the
attached Table.

LAB REPORT: To be prepared for each laboratory session. Submit the handwritten lab
report at the assigned dates (please see Table).

LAB REPORT FORMAT: MUST BE HAND-WRITTEN and NEAT. Points will be

deducted from late reports. Late reports will not be accepted after the graded
reports for that experiment have been handed back to the class.

1. Title of the Experiment

2. Introduction, Objective and principle: (Total should not exceed more than 1 page)
The purpose of the experiment should be clearly stated in no more than five lines. You
may draw some figures if needed.

3. Reagents and Procedure (one page)

4. Results and Calculations: How you prepared your solutions/buffer (show calculation
involved in making the mentioned concentration). Also write your observations which you
note while doing the experiment.( 2 pages)

5. Discussion and Conclusions -This section should include your interpretations of the
data and draw conclusions. You should connect your results with those that were
expected, to those of others and/or to what is already known. Thus, you should expect
to use outside references in writing your discussion. If you obtain unexpected results,
provide possible explanations. This section should be the most detailed and include
interpretation of ALL of your data. The discussion should be no longer than 2 pages.

Answers to the questions given in the time-table for each lab should be submitted
along with the lab report.

GRADING POLICY:
There are 7 labs each carries 10 point for a total of 70% of the grade.
Student must write the pre-lab section and bring it before start of the lab to the TA
and get it signed. The pre-lab section should clearly indicate 1) title of the experiment
2) introduction and objective and 3) reagents and procedure.
Bringing the pre-lab section and getting it initialed by the TA is very important for
getting the pre-lab grade.

10 points of a lab will be counted as: 2 points for the prelab, 6 points for the
Results and Calculations and 2 points for the Discussion and Conclusions.

There will be 6 quizzes carrying 18 % of the grade. The last will be the lab final carrying
10% of the grade.

Quizzes: Quizzes will be given as noted in the timetable. The quizzes will be given at
the lecture portion/lab portion of the course and will be based on the readings and
problems given in the timetable.

Details on Lab Final will be given during the course of the class.

Attendance/Participation: You are expected to participate in each lab session. There

will be no “make-up” labs. Please see the instructor if you must miss lab. Points will
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be deducted if you are not fully participating in the experiment and/or clean-up; for
example, if your partner does most of the work or if you are unable to perform basic lab
techniques.

BE ON TIME!! Each lab period will begin with an important short lecture. In addition,
any changes in procedures are described and required supplies are distributed at the
beginning of the lab period.
Cleanliness, etc. The students in the lab class are responsible for keeping the
equipment and laboratory neat and tidy. Glassware will be cleaned by students in the
laboratory before leaving.

STUDENT RESPONSIBILITIES:
You will need to purchase a copy of the textbook “Modern Experimental Biochemistry”
by Rodney Boyer, Third Edition in the bookstore.
You will need to use a computer and/or a calculator and graph paper to calculate and
analyze data for lab reports.

MSDS: Guidelines for working safely in Chemistry laboratories (MSDS)

Look up for hazards and handling procedures for chemicals you use in the lab.

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 Time Table:

Date Subject of the Laboratory Chapters from Problems/reading Quiz dates/Due

Rodney Boyer dates for lab
report
Jan. 22 Check-in Handout for Lab
1
Jan. 29 Lab 1:Introduction to Handout for Lab pp. 138-139 Quiz 1: Lab 1
electrophoresis: SDS-PAGE 2 1,4,5,8,9 &10
Chapter 4-
pp.111-140
Feb. 5 Lab 2: Using gel filtration to Experiment 3- pp.254 Report of Lab1
study ligand-protein interaction pp.243-256 5,6,9 &10 Quiz 2: Lab 2
(Part I)
Feb. 12 Lab 2: Using gel filtration to Handout for Lab
study ligand-protein interaction 3
(Part II)
Feb. 19 Lab 3: Expression and Experiment 14 pp.428 Report of Lab 2
purification of plasmid DNA (Part and 15- 4,6,7,8,9 & 10 Quiz 3: Lab 3
I) pp. 415-442 pp.440-441
1,2,4,7,8 &10
Feb. 26 Lab 3: The action of Handout for Lab
endonucleases on plasmid DNA 4
(Part II)
Mar 5 Lab 4: In vitro amplification of Quiz 4: Lab 4
DNA by PCR (Part 1) Report of Lab 3
Mar. 12 Lab 4: In vitro amplification of Handout for Lab
DNA by PCR (Part 2) 5

Mar. 19 Spring break

Mar. 26 Lab 5: Kinetic analysis of Experiment 5 pp. 299-300 Quiz 5: Lab 5
tyrosinase (Part I) pp. 279-301 2,7,8,9 &10 Report of Lab 4
April 2 Lab 5: Kinetic analysis of Handout for Lab
tyrosinase (Part II) 6

April 9 Lab 6: Isolation and Experiment 4 pp.55 1 & 4 Quiz 6: Lab 6

Characterization of Bovine milk pp.257-277 pp.207-208 Report of Lab 5
-lactalbumin Part I 1,2,3,4 &9
April 16 Lab 6: Isolation and pp.107 7
Characterization of Bovine milk
-lactalbumin Part II
April 23 Lab 6: Isolation and Handout for Lab pp. 168-169
Characterization of Bovine milk 7 2 &6
-lactalbumin Part III
April 30 Lab 7: Computer Lab Take Home Report of Lab 6
Final Exam
May 7 Check-Out Report of Lab 7
Final exam due

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 CHEM 4242: LABORATORY TECHNIQUES IN BIOCHEMISTRY
Syllabus and Course Information

Lab 1: Introduction to Electrophoresis: Sodium Dodecyl Sulfate Polyacrylamide Gel

Electrophoresis (SDS-PAGE) of proteins
The objectives of the lab are:
 The students will be introduced to electrophoresis; a technique used in
biochemistry and molecular biology to separate proteins according to their size
(length of polypeptide chain or molecular weight).
 The students will learn to determine the molecular weight of unknown proteins by
comparing the mobility of the unknown protein with the mobility of proteins of
known molecular weight.

Lab 2: Using gel filtration to study Ligand-Protein Interactions

This lab has been designed to study the protein-Ligand Interactions- The dynamics of
protein-ligand interactions will be explored with the binding of dye phenol red to bovine
serum albumin. The technique of gel filtration will be used for the molecular separation
of ligand from the complex because the ligand-protein complex often has a molecular
weight much greater than that of the free ligand, which is usually a smaller molecule.
After separation, absorbance measurements will be used to quantify the ligand-protein
complex and free ligand concentration. Data will be analyzed in order to construct the
binding curves.

Lab 3(I): Expression and purification of DNA

The objectives of the lab are:
 To purify plasmid DNA from bacteria. The method involves:
 Growth of the bacterial culture.
 Harvesting and lysis of the bacteria
 Purification of plasmid DNA using anion-exchange resin; and quantification of
the purity and yield of plasmid DNA. The students will be introduced to agarose
gel electrophoresis and to calculate the size and purity of the plasmid DNA.
Lab 3(II): The action of endonucleases (restriction enzymes) on plasmid DNA
Restriction enzymes are used extensively in nucleic acid chemistry. These enzymes
reproducibly cleave DNA at specific sites. This feature provides a convenient way to
break large DNA molecules into smaller fragments for subsequent analysis and
manipulations. It is unlikely that the set of fragments will be the same for two different
DNA molecules, so fragmentation pattern is unique and can be considered a “fingerprint”
of the DNA substrate.

Lab 4: In vitro amplification of DNA by PCR

Students will be introduced to a technique PCR, a technique proved to be a boom to the
molecular biology.
Polymerase chain reaction (PCR) is a process based on a specialized polymerase enzyme,
which can synthesize a complementary strand to a given DNA strand in a mixture
containing the 4 DNA bases and 2 DNA fragments (primers, each about 20 bases long)
flanking the target sequence. The mixture is heated to separate the strands of double-
stranded DNA containing the target sequence and then cooled to allow (1) the primers to
find and bind to their complementary sequences on the separated strands and (2) the
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polymerase to extend the primers into new complementary strands. Repeated heating and
cooling cycles multiply the target DNA exponentially, since each new double strand
separates to become two templates for further synthesis. In about 1 hour, 20 PCR cycles
can amplify the target by about million fold.

Lab 5: Kinetic analysis of Tyrosinase

In this experiment, students will determine the kinetic properties of Tyrosinase, a copper-
containing oxidoreducatase which catalyze the orthohydroxylation of monophenol
substrates and aerobic oxidation of catechols. The enzyme will be assayed by monitoring
the oxidation of 3,4-dihydrophenylalanine (dopa) to red-colored dopachrome. Kinetic
parameters KM and Vmax will be evaluated using Line weaver-Burk or direct linear plots.
Two stereoisomer, L-dopa and D-dopa, will be tested and compared as substrates.
Inhibition of tyrosinase by thiourea and cinnamic acid will be studied. This experiment
has been splitted into two parts.

Lab 6: Isolation and characterization of Bovine milk α-lactalbumin

In this lab, students will learn:
 Isolation of α-lactalbumin from bovine milk.
 Separation of α-latalbumin and β-lactalbumin from other immunoglobulins.
 Separation of α-actalbumin from β-lactalbumin.
 Purification of α -lactalbumin using gel filtration and affinity chromatography.
 Quantification of α-lactalbumin by Bradford assay, SDS-PAGE and UV-Vis
spectroscopy.
 Determination of Molar absorption coefficient of α -lactalbumin.
The lab has been splitted into three parts.

Lab 7: Computer lab (to explore the three-dimensional structure of proteins)

Protein Explorer (http://www.proteinexplorer.org) enables students to visualize
macromolecular structures easily. It is very powerful; in addition to basic
macromolecular visualization capabilities, it offers one-click visualization of interfaces
between moieties ('contacts'), cation-pi interactions and salt bridges, as well as easy-to-
use routines to visualize regions of conservation in three-dimensional protein structures
based on multiple sequence alignments.