Download Fluorescent staining

ISOLATION, SEPARATION AND
DETECTION OF PROTEINS
part I
Michael Jelínek,
Jan Šrámek
Lenka Rossmeislová
Two tasks will be solved at the practise:
1) 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
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
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
METHOD 1:
Fluorescent staining – detection of DNA
UV
DAPI
DNA
DAPI
DNA
„invisible“ in the sample
→ visualization
of detected
molecule after
fluorophore
excitation
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 nongrowing cells ?
Task 2:
Detection of proteins after isolation and separation by SDSPAGE - 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
 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
0.500
y = 0.2286x + 0.0008
R2 = 0.9996
Absorbance (A570 nm)
0.450
0.400
0.350
0.300
0.250
0.200
0.150
0.100
0.050
0.000
0.0
0.2
0.4
0.6
0.8
1.0
1.2
BSA (ug/ul)
1.4
1.6
1.8
2.0
2.2
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