Biochemistry Lab Syllabus (CH
437-52)
Fall 2019
Instructor: Dr.
David Mascotti Class
meets Thursday 8:00-11:50am
Office
location: W207 Class location: DSC W230/221
Office Hours: MWF
9-10 a.m., T 9-12 a.m. & available by appointment.
Communications
Policy: (Read emails that I send you and check my webpage at least once a week
and Canvas when I tell you to do so.)
Telephone:
397-4216
Email: dmascotti@jcu.edu
Webpage: http://webserv.jcu.edu/chemistry/faculty/mascotti/dmascotti/biochem.html
I.
Course Description:
Lab exercises
intended to facilitate understanding of Protein isolation, chromatography,
electrophoresis, quantitative assays, enzyme kinetics, UV/VIS spectroscopy, and
bioinformatics.
II. Goals/Objectives/Learning Outcomes:
General Goals
1. To provide the
student a basic and practical understanding of biochemistry lab techniques and
how they relate to Biochemistry as a whole.
2. To prepare our
students to meet with success in other chemistry and science courses.
3. To prepare our
students for occupations in, and professional programs requiring, a mastery of
General Chemistry such as Graduate School, Medicine, Dentistry, and Pharmacy.
These Goals
address Goals 1-4 of the Departmental Student Learning Goals
1.
Demonstrate
a working knowledge in the sub-disciplines of chemistry where they have
completed coursework.
2.
Apply
their integrative knowledge of chemistry to solve problems.
3.
Demonstrate
competency in the laboratory skills necessary to acquire, analyze and interpret
experimental results
4.
Effectively
communicate scientific information in a variety of forms (written, oral,
mathematical)
These goals, in
turn address the University Goals of “Demonstrate an integrative knowledge of
human and natural worlds”, “Develop habits of critical analysis and aesthetic
appreciation”, “Apply creative and innovative thinking”, “Develop habits of
critical analysis and aesthetic appreciation”, and “Communicate skillfully in
multiple forms of expression”.
Learning
Objectives
1. Learn fundamental
approaches for experimentally investigating biochemical problems
2.
Learn the theoretical foundations for the methods used.
3.
Understand the applicability of the biochemical methods to realistic situations.
Learning
Outcomes
Upon
completion of CH437, students with a passing or above grade will have an
understanding of the following content areas:
1)
Lab
safety
2)
Writing
and reporting results (maintaining lab notebook, lab reports, posters, oral
presentations)
3)
Experimental
design, collection and statistical analysis of data, controls
4)
Reading
the research literature with understanding
5)
Computer
(data analysis, graphing, spreadsheet, literature search, databases)
6)
Preparation
of solutions
7)
Pipetting
liquids
8)
Buffers
and pH
9)
Measurement
of protein and nucleic acid solutions
10) Isolation
and/or characterization of biomolecules (amino acids, peptides, proteins,
enzymes, carbohydrates, lipids, nucleic acids)
11) basic enzymology
12) Centrifugation
13) Microfiltration/membranes
and dialysis
14) Using
commercial kits
15) error and statistical analysis of experimental
data
16) Spectroscopic methods
17) electrophoretic techniques
18) chromatographic separations
19) isolation and characterization of biological
materials
III. Course Expectations:
1. Fundamental Laboratory Approaches for Biochemistry
and Biotechnology, 2nd Edition
by A.J. Ninfa, D.P. Ballou & M. Benore, Wiley Publishing Co., 2009 (recommended)
IV. Grading:
1.
Each student must complete all experiments.
2. Unless announced otherwise, a lab report is required of each
experiment. There is no standard format or length; guidelines will be specified
by instructor. Clear presentation of data, thorough analysis of data, and
neatness are requirements for all reports.
Unless otherwise stated in lab, lab reports are to be handed in one
week after the experiment is completed.
Late reports will be accepted but penalties will apply. No late reports
will be accepted after the last scheduled lab meeting.
3. Each student submits his/her lab notebook at
the end of the semester. Spot checks
will be administered periodically.
4. Short quizzes may be administered at the beginning of each lab
period covering the upcoming experiment.
Those scores would be folded into the evaluation of lab preparation.
5. There will be a
short examination during the last lab period.
Grading
Policy. The grades will reflect the cumulative scores from the
following categories:
Final grades will be assigned on the
basis of:
(a) evaluation of your preparedness to
perform the experiments = 10%
(b) attendance and completion of experiments
= 15%
(c) evaluation of your lab skills and
attitude = 20%
(d) lab report evaluations = 30% (Each report will be evaluated on a scale of
30 points.)
(e) final exam = 15%
(f) evaluation of your lab notebook = 10%
Thus, there are 100 points total. Grades may be curved, but the most stringent
scale that I will use will be <60% = F, 60-67.99% = D, 68-69.99%=D+,
70-72.99%=C-, 73-77.99% = C, 78-79.99%=C+, 80-82.99%=B-, 83-87.99% = B, 88-89.99%=B+,
90-92.99%=A-, and 93+% = A.
Final
Exam. The final exam is mostly cumulative and mandatory. If you know ahead of time that you will miss
this exam with a valid excuse, I will administer an exam before the regularly
scheduled exam.
Absences. Attendance for lab days is obviously mandatory
since you would be unable to finish the lab exercise or write it up.
V. Resources:
Office Hours.
I have an "open door" policy. If I am in my office or lab
(W218), I can usually make time to answer short questions. For more lengthy
discussions I recommend that you come in during regularly scheduled office
hours (see above) or schedule an appointment.
VI. Course Calendar/Topic Outline:
A tentative schedule of experiments is
listed below. Any changes to this
schedule will be announced with sufficient time to adjust your pre-lab
preparation.
A. Most experiments will be performed
individually--without a partner. Experiments to be done with a partner will be
announced in advance.
B. Some
of the experiments require work to be done outside the regular lab periods. A sign-up sheet will be available
to reserve time for you to use the instrument/equipment. The time spent on
these experiments is included in the I4 week schedule. It is not extra work.
PROBABLE
LIST OF EXERCISES/ACTIVITIES
1. Quantitative Protein Assays - quantitative
spectroscopy
2. Isolation of Acid Phosphatase from Wheat
Germ - "salting in/salting out", dialysis, (possibly ion exchange
chromatography) in protein isolation
3. Isolation of Lactate Dehydrogenase from
Chicken Breast - affinity chromatography
4. SDS-PAGE: Sodium Dodecyl Sulfate
Polyacrylamide Gel Electrophoresis - characterization of purity and
estimation of molecular weight
5. Column Chromatography (Gel Permeation and
Ion Exchange) - semiquantitative characterization of
purity, protein separation, and estimation of molecular weight
6. Bioinformatics - sequence analysis software;
Internet tools
7. Protein/Ligand Binding - binding sites in
proteins; quantitative spectroscopy
8. Enzyme Kinetics
9. Laboratory
Lectures on Ancillary Topics - pipetting, centrifugation, pH buffer solutions,
radioactivity, immuno-based protocols, chemiluminescence,
biotin-avidin technology
SCHEDULE
OF EXPERIMENTS
ANY CHANGES WILL BE ANNOUNCED WITH
SUFFICIENT LEAD-TIME FOR ADJUSTMENTS IN YOUR PREPARATION
PERIOD 1: Thursday,
September 5
· Check in to
drawer
· Overview of course and policies (also refer
to lab manual)
- laboratory
notebook
- preparation
of solutions & pipetting techniques
- visible
spectrophotometers
- Excel: computer software for data
analysis and graphing
Exercise 1: Pipetting
& Protein Assays (Bradford and A280 protocols) - Handout
provided
PERIOD 2:
Thursday, September 12
· Exercise 2: Isolation of Acid Phosphatase from Wheat
Germ: Preparation of Crude Extract (Part 1) - Handout provided - and: Acid Phosphatase Isolation - ammonium
sulfate precipitation - Fraction I ®
Fraction II
· Lecture on
Strategies for Protein Isolation (some coverage in the lab manual on pp 157-173)
NOTE: Bradford
assays on Fraction III will be done on your own time outside the regular lab
period.
PERIOD 3: Thursday,
September 19
· Exercise
2 (continued): Acid Phosphatase
Isolation - ammonium sulfate precipitations (Part 2) Fraction II ®
Fraction IV (Part 4)
PERIOD 4: Thursday,
September 26
Activity assays on Fractions I, II, & III
& IV
Bradford assays on Fractions I, II,
& III & IV
PERIOD 5: Thursday,
October 3
· Exercise 3: Isolation of lactate dehydrogenase from
chicken breast via affinity chromatography - Handout provided
The
principles of affinity chromatography are summarized on pp 100-107 of the lab manual.
PERIOD 6: Thursday,
October 10
· Exercise 3 continued: [Protein] and activity
assays and prep samples for next period- Handout provided
PERIOD 7: Thursday,
October 17
- assemble glass plate sandwich; cast running
gel; cast stacking gel; apply protein samples followed by electrophoresis and staining
- destaining and photographs of gels on Tuesday
PERIOD 8: Thursday,
October 24
Exercise 5 : "Half" Period (10-noon) -
Handout provided and pages
337-355
-Bioinformatics and Software on the Web
PERIOD 9: Thursday,
October 31
· Lecture: Western analysis and immunochemistry
· Exercise 6 : SDS-PAGE and electroblotting of albumins
PERIOD 10: Thursday,
November 7
Exercise 6 (continued): Chemiluminescent detection of albumins on
membranes
Lecture: Column
Chromatography
PERIOD 11: Thursday,
November 14
· Exercise 7 : Gel Permeation Column Chromatography
Introduction
to the FPLC, description of exercise and procedure - Handout provided and pages 89-124
PERIOD 12: Thursday,
November 21
Thursday,
November 28 – Thanksgiving Week off
PERIOD 13: Thursday,
December 5
· Exercise 8: Enzyme Kinetics of Tyrosinase –
Handout provided (pages 219-226)
PERIOD
14: Thursday, December 12
· Final Exam, 10% of grade
· Lecture: radioactivity, antibody-based
protocols, biotin-avidin detection protocols, and chemiluminescence
· Clean up and check out
VII.
Additional Policies
1. Academic Honesty:
For
the full JCU policy on academic honesty, please see the University Bulletin, available online and in hard copy.
Academic
honesty, expected of every student, is essential to the process of education
and to upholding high ethical standards. Cheating, including plagiarism,
inappropriate use of technology, or any other kind of unethical behavior, may
subject the student to severe academic penalties, including dismissal.
All
work submitted for evaluation in a course, including tests, term papers, and
computer programs, must represent only the work of the student unless indicated
otherwise.
Material
taken from the work of others must be acknowledged. Materials submitted to
fulfill requirements in one course may not be submitted in another course
without prior approval of the instructor(s).
Concerns
about the propriety of obtaining outside assistance and acknowledging sources
should be addressed to the instructor of the course before the work commences
and as necessary as the work proceeds.
2.
Statement
of Accommodation
In accordance with federal law, if
you have a documented disability you may request accommodations from Student
Accessibility Services (SAS). For more information go to http://sites.jcu.edu/accessibilty. Or you may contact the Director,
Allison West Kaskey at awest@jcu.edu or 216.397.4967.
Please keep in mind that accommodations are not retroactive so it is best to
register at the beginning of each semester. Only accommodations approved by SAS
will be recognized in the classroom. Please contact SAS if you have further
questions.
3.
Statement
on Discrimination, Sexual Harassment, and Bias
John
Carroll University is committed to fostering a learning and working environment
based upon open communication, mutual respect, and ethical and moral values
consistent with Jesuit and Catholic traditions.
The
University seeks to provide an environment that is free of bias,
discrimination, and harassment, including sexual harassment. If you have
experienced sexual harassment/assault/misconduct based upon gender/sex/sexual
orientation, and you share this with a faculty member, the faculty member must
notify the Title IX Coordinator, Eric Butler, who will discuss options with
you. He can be reached at etbutler@jcu.edu or (216) 397-1559. For more
information about your options and resources, please go to http://sites.jcu.edu/title-ix, where you can file an online report.
An option to report anonymously is available.
If
you have experienced bias or discrimination based on race, religion, ethnic or
national origin, disability, age, military or veteran status, genetic
information or any factor protected by law, you are encouraged to report this
via the Bias Reporting System at http://sites.jcu.edu/bias.
For more information about the University’s commitment to diversity and
inclusion, please see: http://sites.jcu.edu/diversity.
VIII.
Other/Additional
This syllabus is a way to ensure that
everyone understands the goals and obligations between the students and the
professor. The professor will adhere to
all policies contained herein, but reserves the right to revise ordering of
topics, timing of topics, and exam dates and give appropriate notice to the
students before changes are made.
General
Information: Keeping a Laboratory Notebook
Each of you should record
information and data taken during the experiment in your own lab notebook. We
will discuss the lab notebooks during the first class period. It is my
intention that you utilize the notebooks as you would in a research or industrial
laboratory. I will review notebooks with you individually during slow times in
the lab period.
All students
will be required to maintain a
laboratory notebook. The notebook will be used
for the recording
of laboratory data
and calculations, and will
be critically important for
writing your lab reports.
The purpose of
a laboratory notebook is to allow anyone with some biochemical knowledge to
understand exactly what you did. You need to record the information in
sufficient detail so as to be able to repeat it, and you must be able to
understand exactly what your results were. You will need good notes to be able
to write your lab reports; in addition, as your understanding of biochemistry
improves, your notebook may allow you to figure out why some parts of your
experiments did not work as expected.
In your
notebook, each experiment should begin with a title, a date, and a statement of
the objective of the planned work. You should also record exactly what you did
at each step (being sure to mention anything that you did that differed from
the information in the Manual). In addition, you should record any numerical
information, such as the weights of reagents used, absorbance readings, enzyme
activities, protein concentrations, and buffer concentrations.
Most experiments
will extend over
several days, and
over several pages
in your notebook. To allow you to
keep track of what you have done, you should include the day’s date at the top
of each page. Including sub-titles for each page may make it easier to keep
track of what you did at each step.
Everything you
do should be recorded directly into your lab notebook in pen. If you make a
mistake, draw a line through it, and write the correction next to the
mistake. (It may
turn out that
the original information
was correct after
all, so do not
obliterate the original information by erasing it, or by removing the page from
your notebook.) Any calculations performed should be written directly into your
book. Hard copies of work done on a computer and printouts from laboratory
instruments should be taped directly into your lab notebook. Writing important information on scrap paper,
and then recording it in your notebook later is not acceptable. If you are writing something while in the
laboratory, you should be writing it directly into your notebook.
At each step in
your experiment (after each assay or measurement), in addition to the results,
record your thoughts regarding the experiment and how you think it is
going. Record your mistakes, and your
attempts to rectify them. Record the calculations involved in any type of
data analysis, as well as explanations for both what you did and what you think
it means. A research project is a journey into the unknown; your laboratory
notebook is usually your only guide through the forests of uncertainty.
It is also a
good idea to look over your notebook periodically during the semester, and make
notes of things that you do not understand, so that you can ask questions
before the lab reports are due.
Do not say
“well, I will remember what this means”; instead, write it down! Do not say “I
will remember what I was thinking while I did this experiment”; instead, write
it down! If you use your lab notebook properly, you will find that writing your
lab reports is much easier, and you will be developing good habits for the
future.
Lab Reports
(total 30 points each)
Tables should be numbered consecutively and
consist of a row and column format, with a title at the top of the table.
Tables should be designed for ease and clarity of reading.