Methods of Studying Cells

8/6/2019 Methods of Studying Cells

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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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Methods of Studying Cells

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