ISOLATION, SEPARATION AND DETECTION OF PROTEINS part I Michael Jelínek, Jan Šrámek Lenka Rossmeislová

Two tasks will be solved at the practise: Detection of proteins and DNA in cancer cells - fluorescent stainning 2) Isolation, separation and following detection of proteins - SDS-PAGE (Sodium DodecylSulfate PolyAcrylamide GelElectrophoresis)

Task 1: Fluorescent staining of microfilaments and DNA actin: phaloidin conjugated with fluorofore TRITC - red signal DNA: DAPI - blue signal used cells - cell line MCF-7 (breast cancer cells) used cytostatic - taxan paclitaxel

Fluorescent staining - principle fluorophor Molecule capable of signal production → visualization of detected molecule Molecule that interacts specifically with the detected molecule Detected molecule „invisible“ in the sample

Fluorescent staining - detection of microfilaments Faloidin poison from mushroom Amanita phalloides binds specifically to microfilaments (F-actin) and blocks their depolymerization (mechanisms of its cytotoxic action) fluorophore TRITC is excited by green light and emits red light

METHOD 1: Fluorescent staining – detection of microfilaments TRITC → visualization of detected molecule after fluorophore excitation phalloidin F-actin „invisible“ in the sample

Fluorescent staining - detection of DNA DAPI intercalates into small groove in dsDNA, this bond increases excitation properties of DAPI after excitation by UV light it starts to emit blue light, free DAPI shines considerable less - it is not necessary to wash

DAPI METHOD 1: Fluorescent staining – detection of DNA DNA DAPI DNA UV → visualization of detected molecule after fluorophore excitation DAPI DNA „invisible“ in the sample

Fixation - first step in sample preparation Prevent degradation and autolysis of tissue and cells Purpose  to preserve the biological material (tissue or cells) as close to its natural state as possible Formaldehyd (= formalin) creates covalent chemical bonds between proteins in tissue anchors soluble proteins to the cytoskeleton

Protocol: fixation of the cells using solution of formaldehyde in PBS (phosphate buffered saline) removal of formaldehyde solution from the cells by repeated wash with PBS incubation with phalloidin-TRITC removal of unbound phalloidin-TRITC by repeated wash with PBS staining with DAPI observation under fluorescent microscope

Fluorescent staining of microfilament Contractile ring

Fluorescent staining of microfilament and DNA How do nuclei and shape of cells differ in growing and non-growing cells ?

Task 2: Detection of proteins after isolation and separation by SDS-PAGE - today first part Tested samples: chicken muscle, BSA solution, milk Isolation of proteins from tissue: first step is disintegration of tissue and cells Chemical (used in our experiment) mechanical ultrasound

Protocol: Isolation of proteins Determination of protein concentration Transfer of tissues and proteins into microtube Disintegration of cells and releasing of proteins by lysis buffer containing SDS (sodium dodecylsulfát) Separation of protein suspension from non - lysed rests by centrifugation Determination of protein concentration By the Bradford method using BSA (bovine serum albumine) as a standard for calibration curve construction

Principle of the Bradford assay assay based on absorbance shift of Bradford reagent that occurs after its binding to proteins Bradford reagent contains Coomassie Brilliant Blue dye - binds to basic and aromatic amino acid residues (ARG, PHE, TRY and PRO) when the dye binds to proteins, it is converted to blue color detection at 595 nm

Calibration curve

Separation of proteins by the SDS-PAGE method To be continued... Separation of proteins by the SDS-PAGE method boiling of the samples with sample buffer containing SDS loading the samples containing desired amount of protein onto a polyacrylamide gel separation of proteins by vertical gel electrophoresis Identification of proteins staining of the gel with the separated proteins in Coomassie blue solution detection of actin and other proteins localization in the gel, comparison of actin and myosin expression among tissues