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53146 7&89

1. Conceptual Questions
2. Hands-on Questions
3. Data Interpretation Questions

Lab 1: Pipettes and Microscopy
P-20, P-200, P-1000 1000L = 1mL
How to use a micropipettor
Conversions between mL and L
How to use a stage micrometer and ocular micrometer to estimate the size of an object
Conversion of ocular units to microns/millimeters
Resolution vs Magnification
! Objective lens working distance and magnification (remember: higher
magnification = longer objective lens = shorter working distance)
! Resolution limits of light microscope vs human eye
! Objective lenses and R values: (Remember: higher resolution = smaller R value,
where R represents the minimal distance two objects can be apart in order for
them to be resolved as separate)
How do we introduce contrast in light microscopy (3 ways discussed in lab manual)?
Basic principles underlying fluorescence microscopy (excitation vs emission)

Lab 2: Spectrophotometry
Spectrophotometry basic principles of
The Bradford Assay what does it allow you to measure? What chromophore does it
use and what wavelength of light does it absorb?
Relationship between light absorbance and concentration
! OD = ecl or A = bc : what does each symbol represent?
How can you use BSA standards to construct a standard curve (whats on the x- and y-
axes)?
How can you use a BSA standard curve to estimate the concentration of an unknown
sample?
Making dilutions

Lab 3: Proteins A - Physical Properties of Proteins & Methods of Isolation
Whole blood after centrifugation
! Top 55% - plasma 91% water, 7% proteins, 2% nutrients/electrolytes/hormones
! Middle 1% - buffy coat white blood cells/platelets
! Bottom 44% - red blood cells (most dense)
What does it mean to precipitate a protein from a given liquid sample?
Explain how these two methods of protein precipitation work:
! Salting out
! Organic solvent
What does dielectric constant essentially describe/measure?

When proteins are precipitated in a given sample, and the sample is centrifuged, what
will be in the supernatant and what will be in the pellet?
How could you use spectrophotometry to estimate total protein yield from a given
precipitation experiment? What info would you need to know?


Lab 4: Proteins B Protein Quantification and Gel Electrophoresis
SDS PAGE:
! What does this stand for?
! Explain the purpose of each component (the SDS, the polyacrylamide gel matrix,
the electric current)
! Ez = fv, what does each symbol represent, what does this equation tell you about
the relationship between the charge, shape, and size of a substance and its
migration through an electric field?
! Bromophenol blue vs Coomassie blue (Remember: bromophenol blue tracks
sample migration / progress of electrophoresis but does not bind proteins, so it
doesnt stain them ; Coomassie blue binds proteins and can stain individual
proteins in a gel after electrophoresis)

Lab 5: Enzyme Kinetics
Characteristics of enzymes

Trypsin -Where is it made? Where does it act and what does it do?
.
BAPNA (aka BAPA) Whats this? What was it used for?
P-nitroaniline (aka pNA)- Whats this? What was it used for?
For Michaelis-Menten Plots, which plot enzymatic rate as a function of [substrate], how
would you define /locate
Km -
Vmax
How can spectrophotometry be used to measure enzyme kinetics:
What steps would you need to take?
What is V
0
and how would you calculate its value from your experimental
data?

! Two general types of enzyme inhibitors:
Competitive
Do they bind active site or another site on enzyme?
What is their effect on Km?
What is their effect on Vmax?
Noncompetitive
Do they bind active site or another site on enzyme?
What is their effect on Km?
What is their effect on Vmax?
Can you distinguish between a competitive and noncompetitive inhibitor if given a:
Michaelis-Menten plot? Lineweaver Burke plot?



Lab 6: The Action Potential
Structure of neuron
Membrane potential
! What is this?
! What are 4 membrane transport proteins that play a role in establishing / altering
this?
! Units?
What is an action potential? Describe each of the following features of a typical
neuronal action potential:
Resting potential
Threshold
Depolarization
Repolarization
Hyperpolarization/Refractory Period
Stimulus current
! What the importance of this?
! Units?
Conductance
! What is this?
! Units?
! Shape of Na+ conductance curve?
! Shape of K+ conductance curve?
Neurotoxins and their effects on channels and action potentials:
! TEA
! TTX

Lab 7: Cell communication
Cell communication: 3 steps? What are receptors? Ligands?
Basic function of the autonomic nervous system
Regulation of heart rate by the autonomic nervous system
! Sympathetic pathways (neurotransmitter used? Autonomic ganglion receptor
used?)
! Parasympathetic pathways (neurotransmitter used? Autonomic ganglion
receptor used?)

Heart Conduction system: Which component generates the action potential?
Two features of cardiac muscle cells that enable the heart conduction system to
work:
! They are connective: connected to one another via gap junctions (allow
free flow of ions/action potentials from one cell to another)

They are autorhythmic: can spontaneously contract at a regular rhythm
without stimulation from nervous system

Agonist vs Antagonist
Basic features of Daphnia Magna (type of organism, basal heart rate)
.
Lab 8: Apoptosis
Apoptosis
! Definition?
! What are some morphological features of apoptosis?
! What are some molecular features of apoptosis (that you studied in lab)?
! What does each of the following reagents/assays allow one to measure/assess in a
given apoptosis experiment?
Trypan blue
Eosin/Methylene blue
Annexin V staining
DNA agarose electrophoresis
SDS PAGE and western blotting (what specific proteins might you study
and what general results would you look for each protein studied?)

HL60 cell line what are these cells? What is the origin of these cells?