Pick up a copy of the course
description on bench by the
clock
Biochemistry 452
Dennis T. Brown
Room 346 Polk Hall
Phone 515 5765
E-mail: dennis_brown@ncsu.edu
Graduate TA: Both sections
Gabriela Schroder
gcschrod@ncsu.edu
E-mail
• Major mechanism of communication
– Notify you of changes in schedule and project
– Send updates on experiment results
– Answer your questions
• Keep your mail box clean
– Lecture notes on web as too large to send
– Some Mails may still push you over quota
Lab Text
• BCH 452 “Experimental Biochemistry, by
Hardin-Ascencio-Knopp, Kendall-Hunt Eds.
ISBN 978-1-4652-1311-2. Available at the
University Bookstore or at Amazon.com
(http://www.amazon.com/ExperimentalBiochemistry-HARDIN-CHARLES
Lecture Notes and other info
posted on Biochemistry web
• http://biochem.ncsu.edu/faculty/brown/bro
wn.php
• At top near photo click on “Biochemistry
452 Notes”
• Look at Powerpoints prior to lab
Schedule
• Lab opens at 1:15
– Turn in Pre lab questions & lab reports
– Collect any handouts
• 1.30 lecture
• 2:00 lab exercise
• Finish usually around 5:00
NO MAKE UP LABS
• Late assignments without a valid excuse will not be accepted and
will receive a score of zero.
• Incomplete as a course grade will be awarded only for work not
completed during the course due to conditions deemed by the
instructor to be beyond the reasonable control of the student.
• Attendance to all laboratories is mandatory and unexcused
absences will affect the outcome of the course.
Bch 452
• Will introduce fundamental techniques in
biochemical analysis
• Will teach the principles upon which the
technology depends
• Will make you familiar with theory and
practice of the technology
More Importantly BCH 452
• Will stress proper laboratory technique
– Reproducibility
– Eliminate errors
• Will stress proper record keeping and data
reporting
– Necessary for archival considerations
– For intellectual property
– For trouble shooting
Concept of 452
• Teach techniques in common use in
biochemistry laboratory science
• Try to present situations that exist in a real
laboratory situation
– Move from “exercises” with predictable
outcomes.
– To “experiments” with doubtful outcomes
• Important to understand and explain
anomalous results
My Job
• To be certain that you understand the principles
that the experiments will examine
• To be certain that you understand how the
apparatus employed in the experiment functions
• To be certain that you understand the reason for
each step in the experimental protocol
Lab Safety (Page XXIII)
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Students are expected to observe proper laboratory procedures as
described in the lab manual (Page XXVII)
a. This is a serious aspect of lab science. Go through the list in the
manual.
b. Read each laboratory experiment before you come to class.
c. Follow precautionary statements given in each experiment.
d. Only necessary work materials should be at or on the laboratory bench.
Do no place coats, backpacks, or other personal belongings on your lab
bench; store them in the lab cabinets or on the coat rack. Do not place them
on the floor.
e. Do not eat, drink, smoke or place materials in your mouth while in the
laboratory.
f. Wear appropriate clothing and eye protection while in the laboratory and
experiments are in progress NO OPEN TOE SHOES
g. Report all accidents to the instructor or TA immediately: such as spills,
cuts, burns, or contact between reagents and your body or eyes. Take
appropriate steps in an emergency situation (such as the use of the eye
wash station or emergency shower).
Hazardous materials
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Hazardous reagents
Volatiles
Corrosives
Mutagens
Material Safety Data Sheets (MSDS)
– Important information regarding all chemicals
– Example on page XXV
Model Molecules
• The three major classes of biomolecules
– Carbohydrates
– Proteins
– Nucleic acids
Model Techniques
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Spectrophotometry
Centifugation
Chromatography
Electrophoresis
Biochemical fractionation
PCR
Your Responsibility
• Read the experiment before you come to
lab
– Read pertaining sections of appendix
– Read “Info Boxes”
• Turn in the pre lab exercise
• Have all required materials
• Some of the protocols can be
confusing. Make a flow sheet that
outlines the steps.
Grading
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Lab reports: 130 total points
Pre Lab Questions: 33 points
Exams (2): 25 points each
Total 216 points
A+ 216 - 205
C+
A 204 - 194
C
A- 193 - 184
CB+ 183 - 173
D
B 172 - 162
F
B- 161 - 151
150 - 140
139 - 130
129 - 119
118 - 108
107 - 0
Extra Credit
•
1. An extra 10 points is added to the Protein Purification
and Southern blot Lab reports due to increased write-up
requirements.
•
2. Lab C1: 5 points extra credit for Arrhenius plot and
activation energy calculation (see page 94)
•
3. 7 extra credit points possible for a well-kept lab note
book
Reporting
• Prelab questions
– Clearly indicated (see page 28 for next weeks lab)
– Due at beginning of designated lab
• Ensures that you have read and understand the exercise
• Completed report
– Due at beginning of next exercise
• Report for Lab A1 due at start of lab C1
Laboratory Notebook (ideal) See
Page 3
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No loose pages
Generates copies
All entries dated
Each page dated
Pages signed by a witness attesting to
date (extreme) Why??
• Retention time (7 years) Why ??
Lab Reports (Page 4)
• Should contain
• Statement of purpose
– Why you are doing experiment
– What you wish to determine
• Brief account of theory and design of experiment
– Summary or flow chart
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The raw data
All calculations
Results
Conclusions and interpretation
A form for lab write up in lab
manual
• Title all sections
– Abstract, Discussion, Graph Titles etc.
• Abstract should contain a concise summary of
experiment
– “I investigated the effect of temperature on activity of
the enzyme Stickase by monitoring its ability to
convert stick to broken stick. This was done by
determining the accumulation of broken stick by
spectrophotometry (A260) at temperatures between 10
to 60 degrees C. I found that Stickase converted stick
to broken stick most efficiently at a temperature of
37oC”
Lab Report
• Record all your experimental data
including changes in protocol
• Label co ordinates. Be careful with units,
spelling and grammar
• Explain sources of error
– It is not as important that you get a “correct”
outcome as it is that you can explain your
result.
Remember
• Your lab report is your PERSONAL
representation of experimental results
• Because you work in groups the raw data
produced will be identical however your
report should be distinct.
Things that you should know
Units of Biochemistry
• Decade system
– Moles
– Meters
– Liters
– Grams
– Centigrade
Elementary calculations
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Gram Molecular Weight
Dilutions
Rate equations
“What is the molar concentration of a
solution that is 5 millimolar NaCl”
Significant Figures
• Determines the value of a data set
• Includes estimations
– 3.2 is not the same as 3.20 or 3.200
Presentation of Data
• Data tables
• Graphs
A Table or Graph Should:
• Be easy to understand
• Project a message
• Does not require extensive reference to
text
Experiment
• A solution of compound X is reacted with
reagent Y which causes the production of
a light absorbing product. The amount of
light absorbing product indicates how
much compound X is present. The
sample containing X is divided into three
aliquots and the experiment is repeated
three times at three wavelengths.
Lousy Table
Results of the three experiments
Absorbance
values for reaction
#1 at 360, 420 and
540 nm
Absorbance
values for reaction
#2 at 360, 420 and
540 nm
Absorbance
values for reaction
#3 at 360, 420 and
540 nm
0.875 (360) 0.885 (360) 0.823 (360)
0.253 (420) 0.250 (420) 0.244 (420)
0.164 (540) 0.165 (540) 0.157 (540)
Good Table
Absorbance values for the “experiments” at
360,420 and 540 nm
Trial #
A360
A420
A540
1
0.876
0.253
0.164
2
0.885
0.250
0.163
3
0.825
0.244
0.157
Bar Graph A: Compares Values at
each wavelength, Shows
Reproducible
0.9
0.8
0.7
0.6
0.5
Absorbance
0.4
0.3
0.2
0.1
0
Trial 1
Trial 2
Trial 3
A360
A420
A540
Wavelength
Bar Graph B: Compares absorbance at
different wavelength, shows sensitivity
0.9
0.8
0.7
0.6
0.5
Absorbance
0.4
0.3
0.2
0.1
0
A360
A420
A540
Trial 1
Trial 2
Trial 3
What is an “Experiment”
An example from our research
program
Dr. Raquel Hernandez: Research Associate Professor Biochemistry
Experiment
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Theory
Hypothesis to test theory
Experiment suggested by hypothesis
Design of experiment
Result of experiment
Conclusions and expansion of experament
Translation to practical application
Fact &Theory
• Arthropod borne viruses (ArBoViruses)
– A class of medically important viruses that are
carried in nature by mosquitos
• Dengue virus infects 500million people/year.
3billion at risk (40% worlds population).
• No treatment or cure
• Theory: Arboviruses are insect viruses
which have evolved over millions of years
to infect vertebrates
Hypothesis
• As they evolved to infect vertebrates,
Arboviruses acquired new genetic
information necessary for growth in this
completely different genetic and
biochemical environment
• It should be possible to identify those
genetic elements and remove them
– Produce virus which can grow in insects but
not vertebrates
Virus Structure as a Target
Outer shell, 2 proteins
Membrane bilayer. Is penetrated
by the two proteins in the
outer shell
Core. Contains multiple copies of
one protein surrounding RNA
Sindbis Virus Envelopment
E2-E1 Trimer
Mammalian and insect membranes
• Insect membranes
have no cholesterol
• Cholesterol makes
membranes viscous,
ion impermeable and
thick
• Insect membranes
are 4 nm thinner than
vertebrate
• Membrane protein
must span both insect
and mammalian
membrane
Vertebrate
Wild Type
Insect
Deletion Mutant
Sindbis Virus Glycoproteins
Systematically remove
amino acids from
transmembrane domain
E2-Capsid Binding
Domain
E2 Transmembrane Mutants
VYTILAVASATVAMMIGVTVAVLCAC Wild Type (TM26)
VYTILAVASATVAMMIGVTVAVLCAC
TM25(D379M)
VYTILAVASATVAMMIGVTVAVLCAC
TM24
VYTILAVASATVAMMIGVTVAVLCAC
TM23
VYTILAVASATVAMMIGVTVAVLCAC
TM22
VYTILAVASATVAMMIGVTVAVLCAC
TM21
VYTILAVASATVAMMIGVTVAVLCAC
TM20
VYTILAVASATVAMMIGVTVAVLCAC
TM19
VYTILAVASATVAMMIGVTVAVLCAC
TM18
VYTILAVASATVAMMIGVTVAVLCAC
TM17
VYTILAVASATVAMMIGVTVAVLCAC
TM16
VYTILAVASATVAMMIGVTVAVLCAC
TM14
VYTILAVASATVAMMIGVTVAVLCAC
TM12
VYTILAVASATVAMMIGVTVAVLCAC
TM10
VYTILAVASATVAMMIGVTVAVLCAC
TM8
Hernandez et al. J Virol. 2003 Dec;77(23):12710-9.
Growth of TM mutants in mammalian
and insect cells
1.00E+10
1.00E+09
1.00E+08
1.00E+07
1.00E+06
1.00E+05
1.00E+04
1.00E+03
1.00E+02
1.00E+01
1.00E+00
20
25
HR
SV
Y4
TM
24
23
22
21
20
19
18
17
16
14
12
10
8
TM16 & TM17 are host restricted mutants
TM
TM
TM
TM
TM
TM
TM
TM
TM
TM
TM
TM
TM
Mutant
Conclusions
• Shortening transmembrane domain crates
a host range (HR) mutation
– Restricts efficient growth to insect cells
• Suggests approach for making vaccines
for arboviruses
– Use HR mutants as vaccines
Vaccination of mice with TM16 and 17
GROUP
(12 Mice)
VIRUS
s.c.
DOSE
PFU
MORBIDITY
1
Mock
(buffer)
NA
0%
2
TM16
106
3
TM16
UV
4
5
MORTALITY
CHALLENGE
(14 days)
MORBIDITY
MORTALITY
72 hr POST
CHALLANGE
72hr POST
CHALLENGE
PROTECTION
Morbidity/
mortality
0%
SAAR86 1000
pfu i.c.
11/12
5/12
8.3%/58%
0%
0%
SAAR86 1000
pfu i.c.
8/12
6/12
33%/50%
NA
0%
0%
SAAR86 1000
pfu i.c.
11/12
4/12
8.3%/66%
TM17
106
0%
0%
SAAR86 1000
pfu i.c.
0
0
100%/100%
TM17
UV
NA
0%
0%
SAAR86 1000
pfu i.c.
10/12
5/12
16%/58%
 TM 17 provided complete protection from challenge
Patent Strategy
US Patent No. 6,589,533
Issued July 8, 2003
"Genetically-engineered alphaviruses, flaviviruses, and
bunyaviruses with modified envelope transmembrane
glycoproteins and altered host-range phenotype"
US Patent No. 6,306, 401
Issued October 23, 2001
"Genetically-engineered sindbis virus with modified E2
Glycoprotein and altered host-range phenotype"
US Patent No. 7,335,363
Issued February 2008
"Membrane Virus Host-Range Mutations and their use as
Vaccine Substrates"
Three other patents pending
This technology
• Is a vaccine platform
– All Arboviruses have the same
transmembrane domain
– This approach should work for all 700 known
arboviruses
• Create a company to move the technology
to the marketplace
– Originally “Arbovax”
– Now “Vacunas”
Products
• Vaccine for Dengue Fever
– Developed for all 4 serotypes
– Very successful non humane primate trial
• 100% Sero-conversion to all 4 serotypes
– Ready for phase one human clinical trials
• Approved by Food and Drug Administration
• Vaccine for Chikungunya Fever
– Successful trials in mice
– Ready for non human primate trials
• Vaccine for West Nile Fever
– Host range mutants made
– Ready for trials in mice
Translational Science
• From Theory (basic Science) to Medical
Product (application)
• Strategy will work for any of the 700
known arboviruses
– 150 are known to cause human disease
– Some cause disease in animals of agricultural
importance
Next Exercise
• Carbohydrates and standard curve
– Section A1 in lab manual (page 26)
– Read carefully prior to coming to lab.
– Do the pre lab questions turn them in on the
26th-27th
Benchtop Protocols
• Page 387
• The benchtop protocol summarizes step
be step the days experiment. It is more
convenient than having the entire manual
open on your bench
DVD
• The DVD shows step by step each part of
the experimental protocol, watching it will
help prepare you for the days exercise