Transcript Lesson 1: Making Polydiacetylene (PDA) - the legleiter lab

Biophysical Chemistry Unit
for High School Chemistry:
Alzheimer’s Disease Study with
Polydiacetylene Assay
By: Sherry Finkel and Elizabeth Yates
Lesson 1:
Background information on
Beta-amyloid (Aβ)
and Alzheimer’s Disease
Alzheimer’s Disease
• Alzheimer’s disease is a neurodegenerative disease.
– Most common form of dementia.
– Causes severe memory loss and brain function.
• What causes memory loss?
– Deposits within the brain cause damage to neurons.
• Neuritic plaques: Ab
– Located extracellularly (on the outside of the neuron)
• Neurofibrillary tangles: tau
– Located intracellularly (on the inside of the neuron)
Researchers are
interested in studying
the proteins that
cause the brain
deposits so that we
may find a cure.
Write down 3 differences you have
observed between the healthy and
Alzheimer's brain
Assignment for tomorrow: Read this article
Fill out this log on a separate sheet of paper. We will
define the words in class.
Reading Log
Words I did not know:
Definitions:
In-Class Discussion
What is our hypothesis
about Aβ and
Alzheimer’s Disease?
Ideas on how can we test
this hypothesis?
Lesson 2:
Hypothesis Testing
Making Polydiacetylene (PDA)
Formulating our hypothesis
• What have we learned about Alzheimer’s disease?
– Ab deposits on the brain damage the neuron.
• Therefore, causing memory loss.
• Types of Ab aggregates: oligomers, protofibrils, fibrils, and plaques
• Let’s come up with our hypothesis using what we know!
– We believe that…
Certain Aβ aggregates play a larger role in Alzheimer’s
disease than others.
• Great hypothesis, but what is a hypothesis we can test?
– It must be more specific!
What are we missing?
• We must know what procedures are available
to test our hypothesis.
• What are our ideas on how to test our
hypothesis?
To test our hypothesis: use an assay
• A colorimetric ASSAY has been developed to test
Ab aggregate interaction with a model cell
surface.
• What is an assay?
– a standard test used in the sciences
• What do you mean by colorimetric?
– See a visible color change when the desired reaction
occurs.
What is our assay made of?
• Polydiacetylene (PDA) is a color indicator.
– When the desired reaction occurs, it changes colors
from blue to red.
• Color change can be measured quantitatively
with a machine that records light absorbance
measurements.
– This process is called spectrophotometry.
– *More information on Spectrophotometry
• The PDA molecule has a hydrophilic head and a
hydrophobic tail.
• These features help PDA bind to lipid bilayers.
How does the assay work?
• PDA is mixed with lipid
extract to make
vesicles.
• These vesicles are
brillant blue to start and
will show a colorimetric
response depending on
the reaction.
Colorimetric Response
Aβ aggregates are added to the solution.
The aggregates can respond it three
ways:
1. They do not interact with the lipid
bilayer, leaving the PDA blue .
2. They interact at the surface of the
lipid bilayer, turning the PDA from blue
to red.
3. They insert themselves into the
bilayer, turning PDA from blue to
purple.
Green: Aβ aggregate
Grey: Lipid bilayer
Blue: PDA
Red: PDA after color change
Calculations
• Colorimetric Response is also known as %CR
• %CR is calculated using these formulas:
PB = A640/(A640 + A500)
%CR = (PB0 -PB1)/PB0 x 100%
– PB is the ratio of blue reflected light to total reflected light
– PB0 is the control, meaning no Aβ is added to the PDA lipid
vesicles.
• No color change should occur in the control.
• PB1 is the sample with Aβ.
In-Class Discussion
• Read and re-read this
lesson.
• In class, you will write
a paragraph describing
PDA and %CR in your
own words
This was a lot of
information! We will
make sense of it in class.
Lesson 3:
Putting it Together
New Hypothesis
• New, testable hypothesis:
• We believe that…
We will see a higher colorimetric response from
certain aggregates of Ab than others.
Aβ Fragments
• Fragments of Aβ are made of different amino acid sequences and sizes.
• For our assay, we will test: 7 fragments of the Aβ protein.
Can you guess what this picture shows?
Atomic Force Microscope
The images from the
last slide were taken
using an AFM capable
of measuring in
nanometers.
1nm=0.000000001m
An Atomic Force Microscope Diagram.
We will discuss how it works in class.
Aβ Aggregates
• A lipid bilayer was exposed to various Aβ fragments and
observed to see if they formed aggregates.
• Any aggregates on
the surface of the
image appear in
white or black
patterns.
How large is each image?
• These images can
help us predict which
fragment will show
the greatest
colorimetric
response.
Make your prediction
• Which or fragment(s) do you think will have
the highest colorimetric responses?
• Why? Write down your rationale (reasoning
or justification)
Lesson 4:
Methods
Experiment modifications
• We will be performing a modified version of the
research experiment from the Legleiter Lab at
West Virginia University in class.
• We will make the following equipment
substitutions:
– PDA: Bromothymol Blue
– Aβ: Unknown concentrations of Acetic Acid labeled
as each Aβ fragment.
Methods
• You and your group members will add 1mL of
each “fragment” (unknown Acetic Acid dilution)
to 9mL of PDA (Bromothymol Blue).
• You will allow the solution to sit for 5 minutes
before using the spectrophotometer (review lesson
2 on using spectrophotometer).
• You will record your results in Microsoft Excel,
where we will calculate %CR.
In-Class Tomorrow
Remember to wear:
• Pants
• Close-toed shoes
• Hair tie for long hair
Lesson 5:
Data Analysis
Legleiter Lab Aβ Data
Legleiter Lab performed a similar experiment to us.
What do you think? Write down your observations.
Can you identify the positive and negative controls?
Legleiter Lab Aβ Data
What do you think? Describe this graph in your own
words. What does it tell you?
Putting it Together
• What do the graph and image show you?
• What can you determine from the graphs
• Draw your conclusions from the Legleiter Lab
data.
Supplemental Assignment: Statistics
• We know that Aβ 22-35 and Aβ 1-40 showed
the greatest colorimetric responses.
• Can we really assume that these results will be
repeatable?
• The only way we can assume is with statistics.
Innocent until Proven Guilty
• We must be 95% sure that our results will hold
the same relationships if the experiment is
repeated.
• We can only be sure if we perform a
probability test, called a t-test, which gives us
a probability value.
The Probability That Our Results
are Not Repeatable…
• The Probability value (called a p-value) is the
probability that our results cannot be repeated.
(Read aloud 5 times until you memorize it!)
• So, the lower the p-value, the more likely your
results are statistically significant.
• We are looking for a low p-value!
P<0.05
• If p is less than 0.05, then there is less than a
5% (0.05x100) likelihood that our results are
due to chance!
• There is a 95% chance that our results are
repeatable! Small p-values=statistical
significance.
Why p<0.05?
• We can never be 100% sure in science.
• We are willing to accept results that are highly
probable in order to move science forward.
In-Class Discussion
• We will work on:
– Statistics:
• t-tests
• analyzing p-values
– Graphing
• Data analysis
• Different plots
• R2 values