HISTOCHEMISTRY- the application of physical or chemical methods of analysis to identify and localize the chemical substances present in their

normal sites in cells/tissues.  CARBOHYDRATES: PAS technique identifies a number of polysaccharides and carbohydrate-containing compounds  LIPIDS: Sudan IV and Sudan black confer red and black colors on lipids  DNA: presence is detected by Feulgen reaction

PROTEIN ISOLATION
Proteins can be separated from other cell components and from one another on the basis of differences in their physical 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 by 10% 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 of their 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.

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.

SDS-polyacrylamide gel electrophoresis

Loading the wells for protein isolation (those hands look familiar!)

...\..\..\genetics\videos\gelelectrophoresis.exe

Use of MASS 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 profile matches these values.

Tryptic peptides are first separated based on mass within a mass spectrometer. Each peptide is then further fragmented primarily by cleaving its peptide bonds. Repeated applications of determining mass differences yield protein partial amino acid sequence.

ISOELECTRIC FOCUSING. At low pH, the carboxylic acid groups of proteins tend to be uncharged and their nitrogen-containing basic groups fully charged giving most proteins a net positive charge. At high pH, he carboxylic acid groups are negatively charged and the basic groups 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 the appropriate direction, each protein species present migrates until it forms a sharp band at its isoelectric pH.

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.

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 ionexchange matrix depends on both the ionic strength and the pH 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.

ASSAYS FOR DETECTING AND QUANTIFYING PROTEINS

1.Staining. All proteins will stain the same color but the color intensity is proportional to the protein concentration. 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 of fluorescence, enzymes or chromogenic substrates, and colored probes (gold). Some probes can detect and measure rapidly changing intracellular ion concentrations inside cells.

5. Antibodies are powerful reagents used to detect, quantify, and isolate proteins. They are used in affinity chromatography and combined with gel electrophoresis 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 be present 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).

Combined methods for protein isolation, detection, and purification.

Shown are schematic depictions of gels for the starting mixture of proteins (lane 1) and samples taken after each of several purification steps. In the first step, salt fractionation, proteins that precipitated with a certain amount of salt were redissolved; electrophoresis of this sample (lane 2) shows that it contains fewer proteins than the original mixture. The sample then was subjected in succession to three types of column chromatography that separate proteins by electrical charge, size, or binding affinity for a particular small molecule The final preparation is quite pure, as can be seen from the appearance of just one protein band in lane 5.

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 may be tagged with a label that can be visualized with a light or electron microscope. The label can be a fluorescent compound, an enzyme such as peroxidase, a gold particle, or a radioactive 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.

AUTORADIOGRAPHY
 Radioisotopes are taken up selectively by cells to be studied  Exposure of photographic film to their emitted radiation reveal presence of such isotopes in the vicinity of these target cells  Silver bromide crystals in emulsion detect radiation, that reduce them to visible black granules.

Pulse-chase autoradiography, Pancreatic B cells were fed with 3Hleucine 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 its position is revealed by the black silver grains in the photographic emulsion. 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.

 Useful in:  Mapping anatomical location of labelled ligands to visualize and quantify receptors in tissue  Studying 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

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.

 These techniques stain various enzymes within cells and tissues by making use of the enzyme activity 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 the presence 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

 Antigen-antibody reactions are highaffinity interactions  It localizes in tissues the following: a.antigen-antibody reactions b.segments of NA (hybridization) c. specific carbohydrate moieties (lectin-binding) d. macromolecules (e.g. phalloidin interacts with actin in microfilaments).

1.Direct method - marker conjugated directly to the antibody that binds to the molecule we are interested 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).

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 protein x. (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.

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) First step 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 (for fluorescence 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).

Photomicrograph of a section of small intestine in which an antibody against the enzyme lysozyme was applied to demonstrate lysosomes in macrophages and Paneth cells. The brown color results from the reaction done to show peroxidase, which was linked to the secondary antibody.

Medical applications

The technique of coupling a tumor cell with the antigenantibody complex has allowed the production of monoclonal antibodies capable of treating specific disorders.

http://highered.mcgraw-hill.com/olc/dl/120110/micro43.swf

Hybridoma cells are widely used to produce unlimited quantities of uniform monoclonal antibodies which are also used to detect and purify proteins.

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 is detected 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 test then proceeds in the same way as the antibody ELISA.

IMMUNOPRECIPITATION Live specimen is incubated in radioactive amino acids Total proteins are extracted and incubated with specific antibody Antibody will bind to its target protein and form an immune complex, Antigen-Antibody complex is incubated with protein A (bacterial protein that binds tightly to IgG-type antibodies) The bound antibody and target protein are run on a protein gel, and the radioactive band of target protein is visualized

Applications of Immunoprecipitation: Determination of the molecular weight and quantity of immunoprecipitated protein; assess for protein-protein interactions, done by immunoprecipitation for one protein, and then blotting for another protein; quantification of rate of synthesis of a protein in cells by determining the quantity of radio-labeled protein made during a specific amount of time; concentrate proteins that are otherwise difficult to detect.

SIGNAL AMPLIFICATION: IMMUNOGOLD

At the top is a thin section of a yeast mitotic spindle showing spindle microtubules that cross the nucleus, connecting at each end to spindle pole bodies embedded in the NE. Below are components of a single spindle pole body. Antibodies against 4 different proteins of the spindle pole body are used, together with colloidal gold particles (black dots), to reveal where within the complex structure each protein is located.

Immunogold Labelling of Serotonin Receptors in Suicide Victims Control Suicide

5-HT2A

5-HT2C

In this study, immunogold labelling was used to quantify the density of 5-HT2A and 5-HT2C subtypes of serotonin receptors in the PFC of suicide victims and controls. It was found that in suicide victims, there is a significant increase in 5-HT2A, but not 5-HT2C receptors on pyramidal cells of cortical layer III.

Total proteins of the sample are extracted and separated on a protein gel Proteins are blotted on a membrane incubated with a specific antibody. The bound antibody is then visualized with a 2nd antibody directed against the 1st antibody Complex is modified for easy detection (e.g. radioactive labeling, conjugating with enzymes that produce intensely colored and insoluble reaction products with substrates) After incubation, a colored precipitate will form on the membrane, corresponding to the position and quantity of the target protein in the original sample

Lane 1 is a protein size marker ladder which shows different known sizes of proteins, Lane 3 is a cancer sample & lane 5 is a normal sample. Lanes 3 & 5 are the same size as the 2nd spot in the size ladder from lane 1.

Green Fluorescent Protein (GFP)

 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.

 (A) The upper surface of the leaves of Arabidopsis plants are covered with huge branched single-cell hairs that rise up from the surface of the epidermis. These hairs, or trichomes, can be imaged in the SEM.  (B) If an Arabidopsis plant is transformed with a DNA sequence coding for talin (an actin-binding protein), fused to a DNA sequence coding for GFP, the fluorescent talin protein produced binds 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.

Lectin Histochemistry Lectins are proteins derived from plant seeds They are membrane-bound carbohydratebinding proteins that bind to specific sequences of cell-surface carbohydrate residues on both glycolipids and glycoproteins in the process of cell-cell adhesion

Fluorescence microscopy of a human skin tissue section (paraffin fixation) with fungal infection. The target carbohydrate subunit chitotriose [(GlcNAc)3] of the pathogenic fungi are specifically bound to lectin from Phytolacca americana-Atto 488 conjugate (green). The nuclei are counterstained with DAPI (blue).

ION-SENSITIVE INDICATORS
 Rapidly changing intracellular ion concentrations can be measured with light-emitting indicators  Their light emission reflects the local concentration of the ion are used to record rapid and transient changes in cytosolic ion concentration.  Some of these indicators are luminescent, while others are fluorescent. Aequorin is a luminescent protein isolated from a marine jellyfish; it emits light in the presence of Ca2+ and responds to changes in Ca2+ concentration in the range of 0.510 M.

Fluorescent Indicator Dyes

They can be introduced to measure the concentrations of specific ions in individual cells or in different parts of a cell. Visualizing intracellular Ca2+
concentrations by using a fluorescent indicator. The intracellular Ca2+ concentration in a single Purkinje cell (from the brain of a guinea pig) was taken with a lowlight 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.

Caged Precursor
The dynamic behavior of many molecules can be followed in a living cell by constructing an inactive caged precursor, which can be introduced into a cell and then activated in a selected region of the cell by a light-stimulated reaction.

Caged molecules. A light-sensitive caged derivative of a molecule

(designated X) can be converted by a flash of UV light to its free, active form. Small molecules such as ATP can be caged in this way. Even ions like Ca2+ can be indirectly caged; in this case a Ca2+-binding chelator is used, which is inactivated by photolysis, thus releasing its Ca2+.

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 three fluorescent markers: rhodamine-labeled tubulin (red) to mark all the microtubules, a blue DNAbinding 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 until activated by ultraviolet light. (B) A beam of UV light is used to uncage the caged-fluoresceinlabeled tubulin locally, mainly just 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-tubulin signal is seen to move toward the left spindle pole, indicating that tubulin is continuously moving poleward even though the spindle (visualized by the red rhodamine-labeled tubulin fluorescence) remains largely unchanged.

X-RAY DIFFRACTION

X-ray crystallography provides

diffraction 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 is scattered (diffracted) in various directions. The intensity of the diffracted waves is recorded on an xray film or with a solid-state electronic detector. (b) X-ray diffraction pattern for a topoisomerase 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