Detection of Detection of proteinsproteinson slide on slide

preparationspreparationsHistochemistryHistochemistry

Methods of detection -Methods of detection -binding the target proteinbinding the target protein

Immunological; primary antibodiesImmunological; primary antibodies secondary secondary antibodiesantibodies

Small molecules; molecules that Small molecules; molecules that bind our bind our

targettarget Proteins; proteins with interactions Proteins; proteins with interactions

with target proteinwith target protein

Methods of visualization - Methods of visualization - marks on the target binding marks on the target binding

moleculemolecule

Fluorescence; small excitable Fluorescence; small excitable fluorescent moleculesfluorescent molecules

Enzyme; enzymes with color reaction Enzyme; enzymes with color reaction after adding substrateafter adding substrate

Methods of visualization - Methods of visualization - marks on the target binding marks on the target binding

moleculemolecule

Fluorescence; small excitable Fluorescence; small excitable fluorescent moleculesfluorescent molecules

Enzyme; enzymes with color reaction Enzyme; enzymes with color reaction after adding substrateafter adding substrate

Methods of visualization - Methods of visualization - marks on the target binding marks on the target binding

moleculemolecule

Fluorescence; small excitable Fluorescence; small excitable fluorescent moleculesfluorescent molecules

Enzyme; enzymes with color reaction Enzyme; enzymes with color reaction after adding substrateafter adding substrate

eg. Alcaline eg. Alcaline phosphatase (AP)phosphatase (AP)

Actin - Major cytoskeletal protein Actin - Major cytoskeletal protein responsible for movementresponsible for movement

Two forms: G-actin-one moleculeTwo forms: G-actin-one molecule F-actin-polymerized F-actin-polymerized

Cooperation with myosinCooperation with myosin Network through cytoplasmNetwork through cytoplasm Actin dynamics is Actin dynamics is

necessary for lifenecessary for life

PhalloidinPhalloidin Poison from Poison from Amanita phalloidesAmanita phalloides Specifically binds F-actinSpecifically binds F-actin Can be linked with small excitable Can be linked with small excitable

molecule without loosing ability to bindmolecule without loosing ability to bind

DAPI - Fluorescent dsDNA probeDAPI - Fluorescent dsDNA probe

Binds dsDNA in the minor groove by Binds dsDNA in the minor groove by intercalationintercalation

That enhances the ability to emit lightThat enhances the ability to emit light Excitation UVExcitation UV Emission in blueEmission in blue

Double stain, Double stain, actin cytoskeleton and DNAactin cytoskeleton and DNA

Merge from phalloidin-TRITC (red) and DAPI (blue)Merge from phalloidin-TRITC (red) and DAPI (blue)

Double stain, Double stain, actin cytoskeleton and DNAactin cytoskeleton and DNA

Phalloidin-TRITC (red) and DAPI (blue) Phalloidin-TRITC (red) and DAPI (blue)