Download Isolation of proteins

SEPARATION AND DETECTION
OF PROTEINS
Part I
Jana Vobořilová,
Anna Kotrbová-Kozak,
Vlasta Fürstová,
Tereza Kopská
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
METHOD 1:
Fluorescent phalloidin conjugate staining
of actin cytoskeleton:
-cells used - NES2Y (human beta-cells of
Langerhans islets)
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
METHOD 2:
Isolation of proteins from different
types of tissues
-tissues used:
*muscle
*heart
*liver
Isolation of proteins from cells:
- first step – desintegration of cells
- desintegration of cells:
* chemical (we use in our experiment)
* mechanical
* physical
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
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
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
- 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
Absorbance 570 nm
Calibration curve
1.000
0.900
0.800
0.700
0.600
0.500
0.400
0.300
0.200
0.100
0.000
0.000
y = 0.2743x + 0.5219
2
R = 0.9977
0.500
1.000
BSA standard (mg/ml)
1.500
Separation of proteins (will be
continued in the second week of the
practice)
-isolated proteins will be separated by the
method SDS-PAGE
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