Transcript Fluorescent staining

ISOLATION, SEPARATION AND
DETECTION OF PROTEINS
part I
Vlasta Němcová,
Lenka Rossmeislová
Michael Jelínek,
Jan Šrámek
Study of cytoskeletal proteins
by 2 methods:
 detection directly in the cells
- fluorescent staining
 detection following isolation and
separation of proteins
- SDS-PAGE
(sodium dodecylsulfate polyacrylamide gel
electrophoresis)
METHOD 1:
Fluorescent staining of microfilaments,
microtubules and DNA
 actin: phalloidin conjugated with TRITC
 tubulin: paclitaxel conjugated with Oregon
Green
 DNA: DAPI
 cells used – cell line NES2Y
(human -cells of Langerhans islets)
METHOD 1:
Fluorescent staining
Molecule
capable
of signal
production
→ visualization of
detected molecule
→ fluorescence 
fluorophore
Detected
molecule
„invisible“ in the
sample
METHOD 1:
Fluorescent staining – detection of
microfilaments
PHALLOIDIN
 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
METHOD 1:
Fluorescent staining – detection of
microtubules
PACLITAXEL
 occurs naturally in
pacific yew tree
(Taxus brevifolia)
 binds specifically to microtubules and stabilizes
them, blocking their depolymerization
 mitotic poison
 used in anticancer therapy (e.g. breast cancer)
 fluorophore Oregon Green is excited by
blue light and emits green light
METHOD 1:
Fluorescent staining – detection of
microtubules
Oregon
Green
→ vizualization of
detected molecule
after fluorophore
excitation
tubulin
„invisible“ in the sample
METHOD 1:
Fluorescent staining – detection of DNA
DAPI
 intercalates into minor groove of dsDNA
 binding to DNA undermines the ability to be
excited
 excited by UV and emits blue light
METHOD 1:
Fluorescent staining – detection of DNA
UV
DAPI
DNA
DAPI
DNA
„invisible“ in the sample
→ visualization
of detected
molecule after
fluorophore
excitation
Fluorescent staining of microtubules
Fluorescent staining of microfilaments
and DNA
Fluorescent staining of microtubules
and DNA
Fluorescent staining of microtubules,
microfilaments and DNA
Fluorescent staining of microfilaments
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
 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 and paclitaxelOregon Green
• removal of unbound phalloidin-TRITC and and
paclitaxel-Oregon Green by repeated wash with PBS
• staining with DAPI
• observation under fluorescent microscope
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
Isolation of proteins from cells and
tissues:
 first step – desintegration of the tissue
and cells
 desintegration (=lysis) of cells
 chemical (we use in our experiment)
 mechanical
 physical
Protocol:
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
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
 colorimetric assay based on absorbance shift
of Bradford reagent that occurs after its binding
to proteins
 Bradford reagent contains Coomassie Brilliant
Blue dye that binds to basic and aromatic
amino acid residues
(arginine (ARG), fenylalanin (PHE), tryptophan
(TRY) a prolin (PRO)
Coomassie Brilliant Blue
 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
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
Separation of proteins by the SDS-PAGE method
(will be continued in the second part of practice)
 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 actin and other proteins localization in the gel,
comparison of actin and myosin expression among tissues