Download Isolation of proteins

SEPARATION AND
DETECTION OF PROTEINS
Part I
Vlasta Němcová,
Michael Jelínek,
Jan Šrámek
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
(sodium dodecylsulfate polyacrylamide gel
electrophoresis)
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)
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)
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 IAlexa 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
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
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
Determination of protein concentration:
 several methods are routinely used
• the Bradford assay (we use in our experiment)
• Lowry assay
• BCA assay (Bicinchoninic assay)
• ultraviolet absorbance assay, etc.
 all of the listed methods rely on the use of a
spectrophotometer (measurement of absorbance)
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)
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
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
Bradford assay
 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
Another methods for protein
determination:
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
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*A280 - 0.76*A260
BCA method
 BCA = bicinchoninic acid
 the peptide bond itself is responsible for color
development
 purple color is detectable at 562 nm