Detection of proteins

Fluorescent stainning of fixed cells

Detection of proteins – examples of clinical

applicationsDetection of :

Antibodies - indirect detection of pathogens, confirmation of immunisation

Mutated proteins - oncologically significant proteins e.g. p53

Characteristic proteins - e.g. detection of tumor origin according to presence of specific proteins

Overexpressed proteins - endocrinology: high amount of some

hormones, oncology: high level of growth factors Unsuffient expression of proteins - endocrinology: level of insuline in

blood, immunology: level of proinflamatory C-peptid

Methods based on detection of proteins

Immunohistochemistry - specific detection of a proteins by antibodies in samples, e.g. tissue sections

Immunocytochemistry - expression level, localization

Immunodetection of proteins on nitrocelulos membrane (after protein electrophoresis and western blot) – expression level, mutations, forms

ELISA (Enzyme Linked ImmunoSorbent Assay) - performed in 96-well plate, enables quantification of detected proteins, e.g.antibodies present in sera...

Flow cytometry - FACS (Fluorescence Activated Cell Sorting)(e.g.hematooncology: detection of specific membrane markers on blood cells of leukemic patients – prognosis, outcome of transplantation )

How can be target protein (=detected protein)

recognized?

1. Another protein interacts with target protein ( DNase I with G-actin, streptavidin with biotin)

2. Small organical molecules - molecules that specifically bind to target (e.g. phalloidin to F-actin, DAPI to DNA)

3. Antibodies, usually primary antibody recognizes target protein and secondary antibody interacts with Fc region of primary antibody = amplification of the signal

Methods of visualization - marks on the target binding

molecule Electron microscopy – metal is bound

Fluorescent detection – a small fluorescent molecule, Fluorophor, e.g. FITC, TRITC, AlexaFluor... is bound to target protein binding molecule

Enzyme-linked detection - target protein binding molecule is covalently linked to an enzyme enabling vizualization after reaction with its substrate (used in Western blot)

chemiluminiscence - horse radish peroxidase (HRP) – cleaves its substrate luminol and light is generated and detected

enzyme catalyzing color reaction after adding substrate – colored unsoluble spot is detected

Metal (gold) labeled antibody

Fluorescence: small excitable fluorescent molecules

Enzymes catalysing visible product after adding substrate

Methods of visualization – fluorescence microscope

DAPI – direct fluorescent dsDNA stain

binds to the minor groove of dsDNA by intercalation this binding enhances the ability to emit light excitation by UV light emission in blue

Actin - major cytoskeletal protein responsible for movement

two forms:

network through cytoplasm

G-actin = globular, one molecule, unpolymerized form of actinF-actin = filamentous, polymerized into fibers

Phalloidin indirect stainning

A poison from mushroom Amanita phalloides specifically binds F-actin can be covalently linked with small excitable molecule

(fluorophore) without losing ability to bind F-actin

in our case, phalloidin is covalently linked with TRITC

TRITC is excited by a wavelength of 540-545nm (green) and emits light of 570-573nm (red)

DNase I indirect stainning

small protein cooperating and specifically binds G-actin in the nucleus can be linked with small excitable molecule without losing ability to

bind G-actin in our preparation DNase I is covalently linked with Alexa Fluor® Alexa Fluor® is a comercial fluorescent dye Alexa Fluor® is excited by a wavelenght of 495nm (blue)

and emits light of 519nm (green)

the function of this interaction is unclear, however since actin-bound DNase I is enzymatically inactive, it is supposed that the DNase-actin complex might be a storage form of DNase I

that prevents damage of the genetic information.

Triple staining, actin cytoskeleton and DNAPhalloidin-TRITC (red), DNase I-Alexa Fluor (green) and DAPI (blue)

Trojité značení,aktinový cytoskelet a DNA

Phalloidin-TRITC (červeně), DNase I-Alexa Fluor (zeleně) a DAPI (modře)