1. SEPARATION AND DETECTION OF PROTEINS Part I Vlasta Němcová, Michael Jelínek, Jan Šrámek

2.  detection directly in the cells - fluorescent staining  detection following isolation and separation of proteins - SDS-PAGE (sodium dodecylsulfate polyacrylamide gel electrophoresis) Identification of actin, microfilamentum structure of the cell cytoskeleton by 2 methods:

3. METHOD 1: Fluorescent staining of F-actin, G-actin and DNA  F-actin: phalloidin conjugated with TRITC  G-actin: DNase I conjugated with Alexa Fluor 488  DNA: DAPI  cells used – cell line NES2Y (human  -cells of Langerhans islets)

4. Fixation – first step of sample preparation  preserve the tissue from decay, either through autolysis or putrefaction  purpose - to preserve the biological material (tissue or cells) as close to its natural state as possible in the process of preparing samples for examination Formaldehyde  creats covalent chemical bonds between proteins in tissue  anchors soluble proteins to the cytoskeleton  tissue becomes more rigid (easier manipulation)

5. 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 and DNase I- Alexa Fluor 488 removal of unbound phalloidin-TRITC and DNase I-Alexa Fluor 488 by repeated wash with PBS staining with DAPI observation under fluorescent microscope

6. METHOD 2: Comparison of actin and myosin expression in different types of tissues - by SDS-PAGE (sodium dodecylsulfate polyacrylamide gel electrophoresis) Isolation of proteins from different tissues:  tissues used:  muscle  heart  liver

7. Isolation of proteins from cells and tissues:  chemical (we use in our experiment)  mechanical  physical  first step – desintegration of the tissue and cells  desintegration (=lysis) of cells

9. Isolation of proteins  transfer of a tissue sample into a tube  desintegration of the tissue by a lysis buffer containing SDS (sodium dodecylsultate)  separation of the lysate containing proteins from tissue fragments by centrifugation  by the Bradford method  using BSA (bovine serum albumine) as a standard for calibration curve construction Determination of protein concentration Protocol:

10. 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 actin and myosin  staining of the gel with the separated proteins in Coomassie blue solution  detection of localization of actin and other proteins in SDS-PAGE, comparison of actin and myosin expression among tissues

11. Determination of protein concentration:  several methods are routinely used  all of the listed methods rely on the use of a spectrophotometer (measurement of absorbance) the Bradford assay (we use in our experiment) Lowry assay BCA assay (Bicinchoninic assay) ultraviolet absorbance assay, etc.

12. Principle of the Bradford method  colorimetric assay based on absorbance shift of Bradford reagent that occurs after its binding to proteins  Bradford reagent contains Coomassie Brilliant Blue G-250 dye that binds to basic and aromatic amino acid residues (arginine (ARG), fenylalanin (PHE), tryptophan (TRY) a prolin (PRO)

13. Coomassie Brilliant Blue G-250  when the dye binds to proteins, it is converted to blue color  the amount of this blue form is detected at 595 nm to quantify the concentration of proteins

14. Calibration curve

15.  preparation of standards for construction of a calibration curve – several samples with known concentration of protein (bovine serum albumine = BSA dissolved in water is routinely used)  dilution of our sample (lysate) with unknown concentration to fit into the concentration range of the calibration curve  incubation of standards and our samples with Bradford reagent  absorbance measurement (A595)  construction of a calibration curve  determination of protein concentration in the lysate using the calibration curve Bradford assay

16. The Lowry assay  based on detection of tyrosine and tryptophan residues  blue color is developed and detectable with a spectrophotometer in the range of 500-750 nm Another methods for protein determination:

17. Ultraviolet absorbance assay  determination of protein concentration by ultraviolet absorption (260 to 280 nm)  depends on the presence of aromatic amino acids in proteins (tyrosine and tryptophan)  [ Protein] (mg/mL) = 1.55*A 280 - 0.76*A 260

18. BCA method  BCA = bicinchoninic acid  the peptide bond itself is responsible for color development  purple color is detectable at 562 nm