1. SEPARATION AND DETECTION OF PROTEINS Part I Jana Vobořilová, Anna Kotrbová-Kozak, Vlasta Fürstová, Tereza Kopská

2. Identification of actin, microfilamentum structure of the cell cytoskeleton by 2 methods: * detection directly in the cells -fluorescent staining * detection following isolation and separation of proteins -SDS-PAGE

3. METHOD 1: Fluorescent phalloidin conjugate staining of actin cytoskeleton: -cells used - NES2Y (human beta-cells of Langerhans islets)

4. Protocol: -fixation of the cells using formaldehyde/PBS solution -removal of formaldehyde solution from the cells by repeated wash with PBS -incubation with phalloidin-TRITC solution -removal of unbound phalloidin-TRITC solution by repeated wash with PBS -staining with DAPI - observation under fluorescence microscope

5. METHOD 2: Isolation of proteins from different types of tissues -tissues used: *muscle *heart *liver

6. Isolation of proteins from cells: - first step – desintegration of cells - desintegration of cells: * chemical (we use in our experiment) * mechanical * physical

8. Isolation of proteins -transfer of a tissue sample into a tube -desintegration of the tissue by a lysis buffer -separation of the proteins from tissue fragments by centrifugation

9. Determination of protein concentration Protein Assay according to Bradford (determination of protein concentration by the Bradford method by construction of a calibration curve for dependance between absorbance and known concentration of protein standards using BSA) -dilution of the BSA standards and a sample of the lysate -incubation with Bradford reagent -absorbance measurement -construction of a calibration curve -determination of concentration of the proteins in the lysate

10. Principle of Bradford method: -based on the binding of Coomassie Brilliant Blue G-250 dye to the proteins and particularly basic and aromatic amino acids residues (hydrophilic arginine (ARG) and the hydrophobic phenylalanine (PHE), tryptophan (TRY), and proline (PRO) (aromatic amino acid residues). As the Coomassie preferentially binds to select amino acids and changes from a cationic (+) state to an anionic (-) one

11. - Coomassie Brilliant Blue G-250 dye exists in three forms: cationic (red), neutral (green) and anionic (blue) -under acidic conditions, the dye is predominantly in the protonated cationic form (red) -when the dye binds to proteins, it is converted to a stable form (blue) -it is this blue form that is detected at 595 nm to quantify the concentration of proteins

12. Calibration curve

13. Separation of proteins (will be continued in the second week of the practice) -isolated proteins will be separated by the method SDS-PAGE

14. Separation of proteins -preparation of the samples in desired concentration -boiling of the samples with sample buffer containing SDS -loading the samples onto polyacrylamide gel -separation of proteins by vertical gel electrophoresis Identification of the actin -staining of the gel with the separated proteins in Coomassie blue solution -detection of localization of actin in SDS-PAGE