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Electrophoresis
Western blotting
ANIKO KELLER-PINTER MD PhD
LUCA MENDLER MD PhD
Electrophoresis
Principles:
•
an analytical method based on movement of charged
particles (proteins, DNA etc.) under the influence of an electric
field
•
velocity of a particle depends on the:
a) size, shape and charge
b) applied voltage
Classification:
I. Gel electrophoresis
- agarose or polyacrylamide gels
- 1D (vertical / horizontal) or 2D
- protein (native / urea / SDS) or DNA/RNA
II. Capillary electrophoresis
III. Microchip electrophoresis
I. Protein gel electrophoresis- general
• agarose or polyacrylamide gels
• 1D (vertical / horizontal) or 2D
• protein (native / urea / SDS) or DNA
Large molecule,
small charge

slow migration
Small molecule,
high charge

fast migration
migration
Separation
I. Protein gel electrophoresis- horizontal
• agarose or polyacrylamide gels
• 1D (vertical / horizontal) or 2D
• protein (native / urea / SDS) or DNA
The figure was found at http://www.mun.ca/biology/desmid/brian/BIOL2250/Week_Three/electro4.jpg
I. Protein gel electrophoresis- vertical
• agarose or polyacrylamide gels
• 1D (vertical / horizontal) or 2D
• protein (native / urea / SDS) or DNA
The figure was found at http://fig.cox.miami.edu/~cmallery/150/protein/page.jpg
I. Protein gel electrophoresis- Native
• agarose or polyacrylamide gels
• 1D (vertical / horizontal) or 2D
• protein (native / urea / SDS) or DNA
Principle:
•
Separates folded proteins and protein complexes by charge,
size and shape
•
Electrophoretic migration occurs because most proteins carry
a net negative charge in alkaline running buffers
Useful for:
1. Examining protein-protein interactions
2. Detecting protein isoforms
I. Protein gel electrophoresis- Urea
• agarose or polyacrylamide gels
• 1D (vertical / horizontal) or 2D
• protein (native / urea / SDS) or DNA
• Separates denatured proteins
by size and charge
• An useful technique to study
protein modifications
migration
•
•
•
agarose or polyacrylamide gels
1D (vertical / horizontal) or 2D
protein (native / urea / SDS) or DNA
SDS: Sodium dodecyl sulfate
I. Gel electrophoresis
-SDS-PAGE
PAGE: Polyacrylamide gel electrophoresis
• As a detergent SDS destroy secondary, tertiary and quarternary structrure
 DENATURING electrophoresis
• Usually, a reducing agent such as dithiothreitol (DTT) is also added to
cleave protein disulfide bonds
SDS
protein
rod shaped protein
• Due to high density of binding of SDS to
proteins, the ratio size/charge is nearly the
same for many SDS denatured proteins

proteins are separated only by size
migration
I. Protein gel electrophoresisTwo dimensional (2D)
• agarose or polyacrylamide gels
• 1D (vertical / horizontal) or 2D
• protein (native / urea / SDS) or DNA
• The first dimension
separates proteins
according to their
native isoelectric point
using isoelectric
focusing (IEF).
• The second
dimension separates
by mass using
ordinary SDS-PAGE.
• 2D PAGE provides the highest resolution for protein analysis and is an
important technique in proteomic research, where resolution of thousands of
proteins on a single gel is sometimes necessary
I. Protein gel electrophoresisTwo dimensional (2D)
• agarose or polyacrylamide gels
• 1D (vertical / horizontal) or 2D
• protein (native / urea / SDS) or DNA
Haemophilus influenzae cell proteins separated by 2D gel electrophoresis.
The basic proteins are to the right of the gel and the acidic proteins to the left.
High molecular weight proteins are to the top of the gel. (Annenberg Media,
Rediscovering Biology)
I. Protein gel electrophoresisVisualisation of proteins
•
Coomassie blue dye
The position (heigth)
of bands indicates
their relative size
Silver staining
Electrophoresis of serum proteins
•
Agarose gel, native electrophoresis
Beta-2
Beta-1
Electrophoresis of serum proteins
Peaks are evaluated by densitometry
60% 3% 9% 12% 16%
The figures are from http://www.sebia-usa.com/products/hyrys2.html
and http://erl.pathology.iupui.edu/LABMED/GENER27.HTM respectivelly (Feb 2007)
Western blotting
Definition:
Western blotting. Principles and methods.
28-9998-97. GE Healthcare Handbooks
Western blotting- Sample preparation
• Use extraxtion
methods that are as
mild as possible
• Extract protein
quickly, on ice if
possible
• Protect the samples
by the use of
protease inhibitors
• Determine total
protein
concentration
Western blotting. Principles and methods.
28-9998-97. GE Healthcare Handbooks
Western blotting- Gel electrophoresis
Western blotting.
Principles and
methods.
28-9998-97. GE
Healthcare
Handbooks
Western blotting- Gel electrophoresis
• Use sample loading buffer (e. g. Laemmli)
• Use molecular weight marker (Mr)
• Reducing or non-reducing conditions (with or
without mercaptoethanol/ antioxidant)
Western blotting. Principles and methods.
28-9998-97. GE Healthcare Handbooks
Western blotting- Transfer (Blotting)
Electrotransfer:
Western blotting. Principles and methods.
28-9998-97. GE Healthcare Handbooks
Western blotting- Transfer (Blotting)
Types:
1. Wet transfer (gel and membrane fully
immersed in transfer buffer)
2. Semi-dry transfer (faster, consumes less
buffer but less efficient!)
Western blotting. Principles and methods.
28-9998-97. GE Healthcare Handbooks
Western blotting- Transfer (Blotting)
Transfer buffers and running conditions:
Western blotting. Principles and methods.
28-9998-97. GE Healthcare Handbooks
Western blotting- Transfer (Blotting)
Membranes:
1. Nitrocellulose
membrane
2. PVDF membrane
Western blotting. Principles and methods.
28-9998-97. GE Healthcare Handbooks
Western blotting- Transfer (Blotting)
Confirmation of
protein transfer
to the membranes:
Staining the membrane with
reversible or irreversible
protein stains
Western blotting. Principles
and methods.
28-9998-97. GE Healthcare
Handbooks
Western blotting- Antibody probing
Blocking:
Western blotting. Principles and methods.
28-9998-97. GE Healthcare Handbooks
Western blotting- Antibody probing
Primary antibodies:
Monoclonal:
Less sensitive
more specific
Polyclonal:
More sensitive
less specific
Western blotting. Principles and methods.
28-9998-97. GE Healthcare Handbooks
Western blotting- Antibody probing
Secondary antibodies:
•
choice depend firstly on the species in which the primary antibody was
produced
•
certain host species may lead to high background change species or
absorb sec. Ab with non-immune serum from the primary Ab species
•
dilution of sec. Ab may range from 1:100-1:500 000- optimization is
needed!
•
choice of enzyme-labeled
antibodies: alkaline phosphatase
(AP), horseradish peroxidase
(HRP)
•
biotinylated sec. Ab: three-layer
system for low abundance targets
Western blotting.
Principles and
methods.
28-9998-97. GE
Healthcare Handbooks
Western blotting- Detection
Based on:
1. Chemiluminescence
(indirect method;
ECL reaction)
Western blotting. Principles
and methods.
28-9998-97. GE Healthcare
Handbooks
Western blotting- Detection
Based on:
2. Fluorescence
(direct method using
fluorophore labelled
sec. Ab)
Western blotting. Principles and methods.
28-9998-97. GE Healthcare Handbooks
Western blotting- Detection
Based on:
3. Chemifluorescence
(indirect method;
ECF reaction)
Western blotting.
Principles and methods.
28-9998-97. GE
Healthcare Handbooks
Western blotting- Imaging
Types:
1. Digital imaging:
CCD camera-based
imager or scanner
2. Chemiluminescence
detection using Xray film
3. (Autoradiography)
4. Colorimetric
detection (HRP
coupled sec Ab,
peroxide and DAB)
Western blotting. Principles and
methods.
28-9998-97. GE Healthcare
Handbooks
CCD: charge-coupled device
Western blotting- Analysis
Types:
1. Qualitative protein analysis: to verify the presence or absence of a
specific protein of interest
2. Quantitative protein analysis: implies a definition of the amount of
protein on a blot either in relative or absolute terms
•
Some important factors should be considered:
•
Sensitivity
•
Linear dynamic range
•
Signal stability
•
In lane normalization
•
Signal-to-noise ratio
Western blotting. Principles and methods.
28-9998-97. GE Healthcare Handbooks
Image analysis software is needed! (ImageQuant, Quantity One)
Western blotting- Analysis
Example:
Western blotting.
Principles and methods.
28-9998-97. GE
Healthcare Handbooks
I. Gel electrophoresis- DNA (RNA)
•
agarose or
polyacrylamide gels
• 1D (vertical / horizontal)
or 2D
• protein (native / urea /
SDS) or DNA
Visualization under UV-light
after staining by ethidium
bromide
•
•
The DNA band of interest can be cut out of the
gel and the DNA extracted
Or DNA (RNA) can be blotted from the gel into
a membrane by Southern Blotting (Northern
Blotting)
II-III. Capillary and microchip
electrophoresis
Advantages:
•
rapid analysis
•
automation
•
low sample and reagent
consumption
•
high reproducibility due
to standardization and
automation
II. Capillary
electrophoresis
•
Separation in capillaries filled
with buffer solution:
 Electrophoresis of serum proteins
 Sequencing of DNA
II. Capillary
electrophoresis
Sequencing of DNA
DNA sequence electropherograms of the NOD2 gene.
(Jane Alfred, Nature Reviews Genetics )
Electrophoresis of
serum proteins
III. Microchip electrophoresis
microchip
•
tiny channels manufactured
in glass or plasctic that
serve as pathways for the
movement of fluid samples
III. Microchip electrophoresis
”Lab-on-a-Chip”:
Rapid analysis of protein, DNA, and RNA
in fluid samples (microfluidics)
„lab-on-a-chip”
III. Microchip electrophoresis
Microfluidics: The use of microfabrication techniques from the IC
industry to fabricate channels, chambers, reactors, and active
components on the size scale of the width of a human hair or
smaller
III. Microchip electrophoresis
Advantages of microfluidics:
•
•
•
•
Sample savings – nL of enzyme, not mL
Faster analyses – can heat, cool small volumes quickly
Integration – combine lots of steps onto a single device
Novel physics – diffusion, surface tension, and surface effects
dominate
– This can actually lead to faster reactions!