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Tutorial Test 1 Notes These study notes cover all the material included on tutorial test 1. They are concise and to the point to help reduce study time while still preparing effectively. Material is presented in the order in which they were introduced in


Department
Biology
Course Code
BIO315H5
Professor
Danton O' Day
Study Guide
Quiz

Page:
of 5
Microscopy
new microscopic techniques are being
developed each year ranging from light-
based to UV-based to electron-based
types of microscopes:
1. Light Microscopy
not general used for living cells
cells/sections of fixed tissues are
prepared, stained & placed on a
slide
used to examine cell shape &
organization of intracellular
components
don’t work for really small things
b/c a single photon is bigger than
the object itself--> can only say if
object is there
2. Phase Contrast Microscopy
good for examining behaviour of
living cells
light waves are shifted--> diff
parts of cell are seen at diff
levels of brightness
can see general localization of
molecules, large organelles &
cellular constituents but need
stains to delineate what they are
gives more detail than DIC but
lots of artifacts
compares 1 photon to the
photon next to it, therefore
slight differences are
exaggerated (halo around
cell)
amplifies differences in
diffraction
3. Differential Interference Contrast
aka DIC
uses a prism & filters to allow
certain wavelengths of oriented
light through to highlight cellular
structures
used for examining living cells
less detail but more accurate;
can look 3D
4. Fluorescence Microscopy
UV light is passed through
fixed/living tissue to examine
cellular fluorescence
requires 2 filters:
1. exciter: only allows specific
wavelength to excite sample
2. barrier: only allows specific
wavelength of fluoresced
light through (from sample)
cells are tagged with fluorescent
molecules
can determine subcellular
localization
if a fluorescently-labelled
antibody is used, then called
immunofluorescence microscopy
DAPI- a nuclear marker; gets into
grooves of DNA & fluoresces
blue under UV
other fluorophores can be used
to mark proteins
can only see 1 fluorophore at a
time--> take pictures & overlap
them
5. Confocal Microscopy
uses laser light source instead of
UV--> more detail
pin-hole: allows light from a
certain plane to pass through &
blocks out-of-focus planes
clearer picture
no fuzziness
smaller pin-hole = clearer &
better
can take images of all the
different slices of the sample &
computer will construct a 3D
image
6. TEM
transmission electron
microscope
slices bombarded with electrons
sections of tissue undergo
special processing & embedding
in plastic before being stained
with heavy metals
electrons pass through tissue;
passage impeded by heavy
metals--> gives impression of
cellular structure
can look at fine detail
highest level of resolution of
cellular & molecular details
staining can be used
7. SEM
scanning electron microscope
can examine whole organisms,
tissues, cells, & molecules
sample typically fixed & coated
with heavy metals (golds) & then
exposed to electrons for
visualization
can also study living tissues &
organisms
Experimental Approaches
rarely does any single approach provide
the complete picture
some approaches:
1. Gel Electrophoresis & Western
Blotting
SDS-PAGE (sodium dodecyl
sulfate polyacrylamide gel
ecetrophoresis) allows
separation of total cellular
extracts to reveal their major
protein constituents as distinct
bands (coats proteins with
negative charge--> run to red)
proteins denature to primary
structure & bind to SDS;
separated in gel by electric field
based on molecular weights
gel is vertical
must stain proteins--> 2 ways:
1. Coomassie blue- stains all
proteins in gel
2. Antibodies- detect specific
proteins
proteins separated on gel can be blotted
onto different membranes & studied
further
can stain the blot with antibodies &
then detect by enzyme reactions or
fluorescence
called western blotting
used to detect & quantify proteins in
cells/tissues
2. 2DE & Proteomics
two dimensional electrophoresis
(2DE)
a gel (first dimension) containing
a pH gradient is run--> separates
proteins based on their
isoelectric point
gel is then laid on a SDS-PAGE
gel (second dimension)-->
separates proteins based on
molecular weight
proteins are then silver stained to
resolve them
2DE allowed resolution of up to
1500 protein spots on one gel
for proteomics, specific protein spots of
interest are cut out & treated in specific
ways to digest & label them
protein fragments are then subjected
to mass spectroscopy to reveal
identities
can compare to databases
3. Pharmacological Approaches
many agents exist that target
specific molecules while others
target types of molecules
some produces naturally, others
produced by pharmaceutical
companies
drug’s effect must be reversible,
specific for the target & dose-
dependent
similar drugs must give the same or
opposite effects
4. Genetic Approaches
mutants have resolved specific
details about role of specific cellular
components
mutants were generated by
treatment with UV, drugs & radiation
& were selected based on defects in
the process of interest
now possible to directly mutate a
gene of interest (knockout or alter it)
when a gene is knocked out, a new
copy of the gene should be inserted
in a rescue mutant to prove that the
defect was only due to the gene in
question
knockout mutants are used to
study the function of a single
protein
normal genes or altered forms can be
put into an expression vector to see what
over-expression does to the cell
different tags can be added to the
vector to the protein is made with a
tag attached
the subcellular location of tagged-
proteins can then be determined by
fluorescence
altering the gene allows scientists to
determine what parts of the
molecule are involved in localizing it
to a specific region or allowing it to
interact with other molecules
SDS-PAGE & Western Blotting
Protein blotting:
once proteins have been separated
by PAGE they are transferred from
the gel matrix to the surface of a
membrane--> western blotting
proteins can be transferred by
placing the PAGE gel onto a
membrane + blotting paper which
draws the proteins onto the
membrane
or can use sideways
electrophoresis:
types of membranes include
nitrocellulose and PVDF
Immunodetection:
transferred proteins probed with
antibodies that will detect the spcific
proteins against which they were
generated--> called immunoblotting
antibodies can be revealed directly
or indirectly:
direct (primary antibody):
a fluorescent dye (fluorescent
isothiocyanate, FITC) is
cross-linked to the antibody
before its added to
membrane--> detect by UV
an enzyme (horseradish
peroxidase, HRP) is attached
to antibody & a single
enzyme reaction shows the
bands that bound to the
antibody