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

2. 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)

3. 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

4. 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

5. 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

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

7. 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

8. 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

9. 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

10. 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

11. Fluorescent staining of microfilament
Contractile ring

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

13. 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

15. 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

16. 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

17. Calibration curve

18. 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