methods of studying cells
TRANSCRIPT
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HISTOCHEMISTRY- the application of physical
or chemical methods of analysis to identify
and localize the chemical substances presentin their normal sites in cells/tissues.
CARBOHYDRATES: PAS technique identifies a
number of polysaccharides
and carbohydrate-containingcompounds
LIPIDS: Sudan IV and Sudan
black confer red and
black colors on lipids
DNA: presence is
detected by Feulgen
reaction
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Proteins can be separated from other cell components and
from one another on the basis of differences in theirphysical and chemical properties.
1. Gel electrophoresis separates proteins on the basis of
their rates of movement in an applied electric field.
SDS polyacrylamide gel electrophoresis can resolve
polypeptide chains differing in molecular weight by10% or less.
2. Centrifugation separates proteins on the basis of their
rates of sedimentation, which are influenced by their
masses and shapes.
3. Chromatography separates proteins on the basis oftheir rates of movement through a column packed with
spherical beads. Proteins differing in mass are
resolved on gel filtration columns; those differing in
charge, on ion exchange columns; and those differing
in ligand-binding properties, on affinity columns.
PROTEIN ISOLATION
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SDS polyacrylamide gel electrophoresis
(left) separates proteins solely on the
basis of their masses.
Two-dimensional gel electrophoresis(right) can separate proteins of similar
mass.
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SDS-polyacrylamide
gel electrophoresis
Loading the wells for
protein isolation (those
hands look familiar!)
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Use ofMASS SPECTROMETRY to
identify proteins and to sequence
peptides. In the first method,
peptide masses are measured and
sequence databases are then
searched to find the gene that
encodes a protein whose
calculated tryptic digest profilematches these values.
Tryptic peptides are first
separated based on mass
within a mass spectrometer.
Each peptide is then further
fragmented primarily bycleaving its peptide bonds.
Repeated applications of
determining mass
differences yield protein
partial amino acid sequence.
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ISOELECTRIC FOCUSING. At low pH, the carboxylic acid groups of
proteins tend to be uncharged and their nitrogen-containing basicgroups fully charged giving most proteins a net positive charge. At high
pH, he carboxylic acid groups are negatively charged and the basicgroups tend to be uncharged, giving most proteins a net negative
charge. At its isoelectric pH, a protein has no net charge since the
positive and negative charges balance. Thus when a tube containing a
fixed pH gradient is subjected to a strong electric field in theappropriate direction, each protein species present migrates until it
forms a sharp band at its isoelectric pH.
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COLUMN CHROMATOGRAPHY
The sample, a mixture of different molecules, is applied to
the top of a cylindrical glass or plastic column filled with a
permeable solid
matrix, such as
cellulose, immersed
in solvent.
A large amount of
solvent is then
pumped slowly
through the column
and collected in
separate tubes as it
emerges from the
bottom.
Because various
components of the sample travel at different rates through
the column, they are fractionated into different tubes.
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ION-EXCHANGE CHROMATOGRAPHY(A) the insoluble matrix carries ionic charges that retard the
movement of molecules of opposite charge. The strength of
the association between the dissolved molecules and the ion-
exchange matrix depends on both the ionic strength and thepH of the solution that is passing down the column, which may
therefore be varied systematically to achieve an effective
separation. (B) In gel-filtration chromatography, the matrix is
inert but porous. (C) Affinity chromatography relies on
antigen-antibody interactions.
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ASSAYS FOR DETECTING AND QUANTIFYING PROTEINS
1.Staining. All proteins will stain the same color but the
color intensity is proportional to the proteinconcentration.
2.Autoradiography. An x-ray film is apposed to the gel
for a certain time and then developed. Radioactive
proteins will appear as dark bands in the film and can
be used as a semi-quantitative technique for
detecting molecules in cells, tissues, or gels.
3.Pulse-chase labeling can determine the intracellular
fate of proteins and other metabolites.
4.Generating amplified signals through the use offluorescence, enzymes or chromogenic substrates,
and colored probes (gold). Some probes can detect
and measure rapidly changing intracellular ion
concentrations inside cells.
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5. Antibodies are powerful reagents used to detect,
quantify, and isolate proteins. They are used in
affinity chromatography and combined with gelelectrophoresis in Western blotting.
6. Immunoblotting. The isolated proteins are
transferred from the gel to a nitrocellulose
membrane. The membrane is incubated with an
antibody made against proteins that may bepresent in the sample.
7. 3-D structures of proteins are obtained by x-ray
crystallography (provides the most detailed
structures but requires protein crystallization),
cryoelectron microscopy (most useful for large
protein complexes, which are difficult to
crystallize), and NMR or nanomagnetic resonance
spectroscopy (only relatively small proteins are
amenable to NMR analysis).
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Shown are schematic
depictions of gels for the
starting mixture of proteins
(lane 1) and samples taken after
each of several purificationsteps. In the first step, salt
fractionation, proteins that
precipitated with a certain
amount of salt were re-
dissolved; electrophoresis of
this sample (lane 2) shows thatit contains fewer proteins than
the original mixture. The sample
then was subjected in
succession to three types of
column chromatography that
separate proteins by electricalcharge, size, or binding affinityfor a particular small molecule
The final preparation is quitepure, as can be seen from the
appearance of just one protein
band in lane 5.
Combined methods for
protein isolation,
detection, and purification.
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Compounds that have affinity
toward another molecule can
be tagged with a label and used
to identify that molecule.
(1) Molecule A has a high and
specific affinity toward a
portion of molecule B.
(2) When A and B are mixed, A
binds to the portion of B it
recognizes. (3) Molecule A maybe tagged with a label that can
be visualized with a light or
electron microscope. The label
can be afluorescent
compound, an enzyme such as
peroxidase, a gold particle, or aradioactive atom.
(4) If molecule B is present in a
cell or extracellular matrix that
is incubated with labeled
molecule A, molecule B can be
detected.
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Radioisotopes are
taken up selectivelyby cells to be studied
Exposure of
photographic film to
their emitted radiationreveal presence of
such isotopes in the
vicinity of these
target cells
Silver bromidecrystals in emulsion
detect radiation, that
reduce them to visible
black granules.
AUTORADIOGRAPHY
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Pulse-chase autoradiography, Pancreatic B cells were fed with 3H-
leucine for 5 minutes (the pulse) followed by excess unlabeled leucine
(the chase). The amino acid is largely incorporated into insulin, which
is destined for secretion. After a 10-minute chase the labeled protein
has moved from the rough ER to the Golgi stacks (A), where itsposition is revealed by the black silver grains in the photographicemulsion. After a further 45-minute chase the labeled protein is found
in electron-dense secretory granules (B). The small round silver grains
seen here are produced by using a special photographic developer.
Experiments similar to this were important in establishing the
intracellular pathway taken by newly synthesized secretory proteins.
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Useful in:
Mapping anatomical location of labelled ligands to
visualize and quantify receptors in tissueStudying sequence and intensity of events occurring
in tissue components
Measuring DNA production (e.g., 3H-thymidine)
Advantages: protocol is simple & easy to follow
Disadvantages:Everything binds to everything (misinterpret results)
There are no biochemical or physiological criteria to
assess the binding specificity (i.e., to determine
whether the binding site really corresponds to an
actual receptor)The presence of a high-affinity labelled receptor
does not necessarily imply that the receptor has
physiological significance
Ligands are not always very specific
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Methods of introducing a membrane-impermeant substance into a cell(A) The substance is injected through a micropipette, either by applying pressure or, if the
substance is electrically charged, by applying a voltage that drives the substance into the cell
as an ionic current (a technique called iontophoresis). (B) The cell membrane is made
transiently permeable to the substance by disrupting the membrane structure with a brief but
intense electric shock (2000 V/cm for 200 sec, for example). (C) Membrane-enclosed vesicles
are loaded with the desired substance and then induced to fuse with the target cells. (D) Gold
particles coated with DNA are used to introduce a novel gene into the nucleus.
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These techniques stain various enzymes within
cells and tissues by making use of the enzymeactivity itself.
The enzyme is made to react with a specific
substrate. The product of this reaction may itself be
visible in the microscope and thus demonstrate thepresence of the enzyme at a specific location,
or the reaction product is
subsequently reacted to form
a visible secondary
reaction product. Examples:
Acid Phosphatase
Gomori-Takamatsu method
Peroxidase DAB method
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Antigen-antibody
reactions are high-affinity interactions
It localizes in tissues
the following:
a.antigen-antibody
reactions
b.segments of NA
(hybridization)
c. specific carbohy-
drate moieties(lectin-binding)
d. macromolecules
(e.g. phalloidin
interacts with actin in microfilaments).
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1.Direct method - marker conjugated directly to the
antibody that binds to the molecule we areinterested in.
2.Indirect method - marker bound to antibody that will
bind to the antibody that binds to the molecule we
are interested in (i.e. GAM - IgG).
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Direct method of immunocytochemistry.
(1) Immunoglobulin molecule (Ig). (2) Production of a
polyclonal antibody. Protein x from a rat is injected into
a rabbit. Several rabbit Igs are produced against proteinx. (3) Labeling the antibody. The rabbit Igs are tagged
with a label. (4) Immunocytochemical reaction. The
rabbit Igs recognize and bind to different parts of
protein x.
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. Indirect method of immunocytochemistry.(1) Production of primary polyclonal antibody. Protein x from a rat is
injected into a rabbit. Several rabbit immunoglobulins (Ig) are produced
against protein x. (2) Production of secondary antibody. Ig from a
nonimmune rabbit is injected into a goat. Goat Igs against rabbit Ig are
produced. The goat Igs are then isolated and tagged with a label. (3) Firststep of immunocytochemical reaction. The rabbit Igs recognize and bind
to different parts of protein x. This detection method is very sensitive.
Commonly used marker molecules include fluorescent dyes (forfluorescence microscopy), the enzyme horseradish peroxidase (for either
light microscopy or EM), colloidal gold spheres (for EM), and the enzymes
alkaline phosphatase or peroxidase (for biochemical detection).
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Photomicrographof
asectionofsmall
intestine inwhichanantibodyagainstthe
enzyme lysozyme
was appliedto
demonstrate
lysosomes inmacrophages and
Panethcells. The
browncolorresults
fromthe reaction
done toshow
peroxidase, which
was linkedtothe
secondaryantibody.
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The
technique of
couplingatumorcell
withthe
antigen-
antibody
complex hasallowedthe
production
of
monoclonal
antibodies
capable of
treating
specific
disorders.
Medicalapplications
http://highered.mcgraw-hill.com/olc/dl/120110/micro43.swf
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Hybridoma
cells are
widelyusedto
produce
unlimited
quantities of
uniform
monoclonal
antibodies
whichare alsousedtodetect
andpurify
proteins.
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The Enzyme-Linked Immunosorbent Assay (ELISA) is
a technique used to detect antibodies or infectious
agents in a sample.
For an antibody ELISA, antigens are stuck onto a plastic surface, a sample is
added and any antibodies for the disease tested for will bind to the antigens.
Next a second antibody with a marker is added and a positive reaction isdetected by the marker changing color when an appropriate substrate is
added. If there are no antibodies in the sample, the second antibody will not
be able to stick and there will be no color change. For an antigen ELISA,
antibodies are bound to a plastic surface, a sample is added and if antigens
from the virus tested for are present, they will stick to the antibodies. This testthen proceeds in the same way as the antibody ELISA.
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The bound antibody and target protein are
run on a protein gel, and the radioactive
band of target protein is visualized
Antigen-Antibody complex is incubated with protein A
(bacterial protein that binds tightly to IgG-type antibodies)
Antibody will bind to its target proteinand form an immune complex,
Total proteins are extracted and
incubated with specific antibody
Live specimen is incubated in radioactive amino acids
IMMUNOPRECIPITATION
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Applications of Immunoprecipitation: Determination of the molecular weightandquantityof immunoprecipitatedprotein; assess for protein-protein
interactions, done byimmunoprecipitation for one protein, andthen blotting
for another protein; quantification of rate of synthesis of a protein in cells by
determiningthe quantityof radio-labeledprotein made duringa specific
amount of time; concentrate proteins that are otherwise difficult to detect.
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Atthe top is athin
sectionofayeast
mitotic spindle showing
spindle microtubules
thatcross the nucleus,
connectingateachendtospindle pole bodies
embeddedinthe NE.
Beloware components
ofasingle spindle pole
body. Antibodies
against4 different
proteins ofthe spindlepole bodyare used,
togetherwithcolloidal
goldparticles (black
dots),torevealwhere
withinthe complex
structure eachprotein
is located.
SIGNAL
AMPLIFICATION:
IMMUNOGOLD
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Control Suicide
5-HT2A
5-HT2C
In this study,
immunogoldlabelling was used
to quantifythedensityof5-HT2A
and 5-HT2Csubtypes of
serotoninreceptors in the
PFC ofsuicidevictims and
controls. Itwas
found that in
suicide victims,there is a
significantincrease in 5-HT2A,
but not5-HT2Creceptors on
pyramidal cells of
cortical layer III.
Immunogold Labelling ofSerotoninReceptors in Suicide Victims
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After incubation, a colored precipitate will form on
the membrane, corresponding to the position and
quantity of the target protein in the original sample
Complex is modified for easy detection (e.g.
radioactive labeling, conjugating with enzymes that
produce intensely colored and insoluble reaction
products with substrates)
The bound antibody is then visualized with a 2nd
antibody directed against the 1stantibody
Total proteins of the sample are extracted
and separated on a protein gel
Proteins are blotted on a membrane
incubated with a specific antibody.
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Lane 1 is aproteinsize markerladderwhichshows
differentknownsizes ofproteins,Lane 3 is acancer
sample & lane 5is anormalsample. Lanes 3 & 5are the
same size as the 2ndspotinthe size ladderfromlane 1.
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GFP is an especially versatile probe that can be attached
to other proteins by genetic manipulation.
Variants have been generated with altered absorption
and emission spectra in the blue-green-yellow range. A
family of related fluorescent proteins has been
discovered in corals, extending the range into the red
region of the spectrum.
Virtually any protein of interest can be
genetically engineered as a GFP-fusion
protein, and then imaged in living cells by
fluorescence microscopy.
Peptide location signal can also be added to
GFP to direct it to a particular cellular
compartment, such as the ER or a mitochondrion,
lighting up these organelles so they can be observed in
the living state. GFP is also used as a reporter molecule
to monitor gene expression.
Green Fluorescent Protein (GFP)
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(A) The upper surface of
the leaves of Arabidopsis
plants are coveredwith
huge branchedsingle-cell
hairs that rise up from the
surface of the epidermis.
These hairs, or trichomes,
can be imagedin the SEM.
(B) If an Arabidopsis plant
is transformedwith a DNA
sequence coding for talin (an actin-binding protein), fused
to a DNA sequence coding for GFP, the fluorescent talin
protein producedbinds to actin filaments in all the living
cells of the transgenic plant.
Confocal microscopy can reveal the dynamics of the
entire actin cytoskeleton of the trichome (green). The red
fluorescence arises from chlorophyl in cells within the leaf
below the epidermis.
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Lectins are proteins derived from plant seeds They are membrane-bound carbohydrate-
binding proteins that bind to specific
sequences of
cell-surfacecarbohydrate
residues on both
glycolipids and
glycoproteins inthe process of
cell-cell adhesion
Lectin HistochemistryLectin Histochemistry
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Rapidly changing intracellular ion
concentrations can be measuredwith light-emitting indicators
Their light emission reflects the
local concentration of the ion are
used to record rapid and transient
changes in cytosolic ionconcentration.
Some of these indicators are
luminescent, while others are
fluorescent. Aequorin is a
luminescent protein isolated from amarine jellyfish; it emits light in the
presence of Ca2+ and responds to
changes in Ca2+ concentration in
the range of 0.510 M.
ION-SENSITIVE INDICATORS
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Visualizing intracellular Ca2+
concentrations by using a
fluorescent indicator.
The intracellular Ca2+
concentration in a singlePurkinje cell (fromthe brain of a
guinea pig)was taken witha low-
light camera and the Ca2+-
sensitive fluorescent indicator
fura-2. The concentration of free
Ca2+ is represented by different
colors, red being the highest and
blue the lowest. The highest Ca2+
levels are present in the
thousands of dendritic branches.
Fluorescent Indicator Dyes
They can be introduced to measure the
concentrations of specific ions in individual cells orin different parts of a cell.
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Caged molecules. A light-sensitive caged derivative ofa molecule(designated X)canbe converted bya flashofUVlightto its free, active
form. Small molecules suchas ATPcanbe caged inthis way. Evenionslike Ca2+ canbe indirectlycaged; inthis case a Ca2+-binding chelator is
used,whichis inactivated byphotolysis,thus releasing its Ca
2+.
Caged Precursor
The dynamic behaviorofmanymolecules canbe followed in
a living cell byconstructing aninactive caged precursor,whichcanbe introduced into a cell and thenactivated ina
selected regionofthe cell bya light-stimulated reaction.
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Determining microtubule flux in the mitotic
spindle with caged fluorescein linked to tubulin(A) A metaphase spindle formed in vitro from an
extract of Xenopus eggs has incorporated threefluorescent markers: rhodamine-labeled tubulin
(red) to mark all the microtubules, a blue DNA-
binding dye that labels the chromosomes, and
caged-fluorescein-labeled tubulin, which is also
incorporated into all the microtubules but is
invisible because it is nonfluorescent untilactivated byultraviolet light. (B) A beam of UV
light is used to uncage the caged-fluorescein-
labeled tubulin locally, mainlyjust to the left
side of the metaphase plate. Over the next few
minutes (after 1.5 minutes in C, after 2.5
minutes in D), the uncaged fluorescein-tubulinsignal is seen to move toward the left spindle
pole, indicating that tubulin is continuously
moving poleward even though the spindle
(visualized bythe red rhodamine-labeled tubulin
fluorescence) remains largelyunchanged.
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X-ray crystallography providesdiffraction data from which the 3D
structure of a protein or nucleic acid
can be determined.(a)Basic components of an x-ray
crystallographic determination.
When a narrow beam of x-rays
strikes a crystal, part of it passes
straight through and the rest isscattered (diffracted)in various
directions.The intensity of the
diffracted waves is recorded on an x-
ray film or with a solid-state
electronic detector.
(b)X-ray diffraction pattern for atopoisomerase crystal collected on a
solid-state detector. From complex
analyses of patterns like this one, the
location of every atom in a protein
can be determined
X-RAY DIFFRACTION
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