Detection of proteins Fluorescent stainning of fixed cells

Detection of proteins – examples of clinical applications Detection 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 actin F-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 nm (green) and emits light of nm (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 DNA Phalloidin-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)