Chem. 453: Biochemical Methods, Fall 2000
Sect. 001G - 1:00-4:50 pm Tuesdays
Phillips 469
Scott Hartsel
P461, 836-4746
Tentative Schedule
Date
Topic
Data collection & data fitting/ Molecular Modeling and
Spartan
Chapter 1,2-NB
T
Sept.
6
13
Reading
La b M a nua l (LM )
Ninfa a nd B a llo u (NB )
SAFETY, etc
Spectroscopic Methods
Peptide sequencing and analysis
RP-HPLC method development for Peptide
Characterization
20
Aspartame–sequence and decomposition
27
Protein Structure, Chromatography and Sequencing and
HPLC
Chapter 2-NB
http://hplc.chem.shu.edu
/NEW/HPLC_Book/
Peptide Sequencing 1LM
Chapter 2-NB
Fitting Enzyme Kinetic Data
Oct.
4
11
The Enzymatic Activity of Alkaline Phosphatase from
the Calf-Fitting Kinetic Data
Semester Project B: Isolation and
Oct.
Characterization of Alkaline Phosphatase from E.
Coli
18
25
Nov. 1
8
15
22
29
Dec.
Day 1:Isolation, shock, AP Assay
Day 2: Heat and NH4SO4 purification, AP Assay,
Prepare resin. HPLC methods.
Enzyme Kinetics 1-LM
Chapter 8,9-NB
Chapter 6,7-NB
SP B:Part A-C
SP B:Part D-G
Day 3:Anion Exchange purification, AP Assay, Protein
Assay
SP B:Part I,H
Day 4:Protein Assay, Yield, SDS electrophoresis prep
SP B:Part I,H
SP B:Part J
Day 5: SDS Electrophoresis/Western Blot
Day 6: pH dependence of AP activity
Day 7:Km & kcat of AP activity
6 ,13
Check-out, finish up
Final
1:00 PM Tues. Dec. 20th
Dates when reports are due will be announced in class.
Enzyme Kinetics1-LM
Texts:
Chem. 453 Lab Manual (Handed Out)
Biochemistry and Biotechnology, Ninfa and Ballou (Textbook Rental)
Required
Materials:
Bound notebook and pen for taking lab notes
A binder for your lab manual
Safety goggles (They must meet ANSI Z87.1-1989 standards (Available for purchase
at the bookstore)
“Sharpie” pen or wax pencil for marking glassware/a glue stick is a good idea
1 Data Collection and Fitting-Unknown
2 Molecular Modeling-Post-lab
3 Aspartame Analysis and Decomposition
4 Enzyme Kinetics of Alkaline Phosphatase from the Calf
5 Isolation and Characterization of Alkaline Phosphatase
6 Final Exam
7 Lab Notebook !!
8 Miscellaneous Assignments, Lab Skills
5
10
50
50
100
25
25
35
Total
300
Grade Scale
(typical)
90-100 A
80-89 B
70-79 C
60-69 D
Lab Reports
Full individual lab reports will be submitted for, Aspartame Structure and
Decomposition, Enzyme Kinetics of Alkaline Phosphatase from the Calf and
Isolation and Characterization of Alkaline Phosphatase from E coli. See the
format below and I will hand out an example. Deadlines will be announced in class
Format for Lab Reports (except for the semester project, and Isolation and Characterization of Alkaline
Phosphatase,which will be discussed in class) WRITE REPORTS IN NARRATIVE FORMAT, NOT
AS OUTLINES OR WITH FRAGMENTARY SENTENCES!!! :
I.
Introduction:
A. Give a brief statement of the purpose of the lab, what you expect to observe, the theory behind
the techniques used and any assumptions used in applying those techniques .
II.
Material and Methods:
A. Give a complete description of the procedures you will be using. Remember, be concise yet
thorough enough that someone could reproduce your experiments.
III.
Results:
A. Present you results in tabular or graphical form .
1. Clearly label your tables and graphs.
B.
In the body of the results section give a concise description of what each table or graph shows.
1. Include any values which were derived from data presented in the tables or graphs and
indicate how they were derived.
IV. Discussion:
A. Give a summary of your results.
B. State whether you observed what you expected to observe.
C. Use the questions asked in the lab manual to help structure your discussion.
V.
Conclusions:
A. Give a brief statement (a couple of sentences) of what conclusions can be drawn from the
experiment.
1. Describe how your results support your conclusions.
2. Discuss whether the outcome was expected and if not, how your assumptions may have impacted
your results.