Electrophoresis Western blotting

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HURO/0901/069/2.3.1 

HU-RO-DOCS 

Electrophoresis 

Western blotting 

ANIKO KELLER-PINTER MD PhD 

LUCA MENDLER MD PhD

Principles: 

Electrophoresis 

• 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 




The figure was found at http://www.mun.ca/biology/desmid/brian/BIOL2250/Week_Three/electro4.jpg

I. Protein gel electrophoresis- vertical 




The figure was found at http://fig.cox.miami.edu/~cmallery/150/protein/page.jpg

I. Protein gel electrophoresis- Native 




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 




• Separates denatured proteins 

by size and charge 

• An useful technique to study 

protein modifications 

migration




I. Gel electrophoresis 

-SDS-PAGE 

SDS: Sodium dodecyl sulfate 

• As a detergent SDS destroy secondary, tertiary and quarternary structrure 

DENATURING electrophoresis 

PAGE: Polyacrylamide gel electrophoresis 

• Usually, a reducing agent such as dithiothreitol (DTT) is also added to 

cleave protein disulfide bonds 

SDS 

protein 

rod shaped protein 

migration 

• 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

• 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. 

I. Protein gel electrophoresis- 

Two dimensional (2D) 




• 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


Two dimensional (2D) 




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)


Visualisation of proteins 

• The position (heigth) 

of bands indicates 

their relative size 

Coomassie blue dye 

Silver staining

Electrophoresis of serum proteins 

• Agarose gel, native electrophoresis 

Beta-2 

Beta-1


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- 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 (M r ) 

• Reducing or non-reducing conditions (with or 

without mercaptoethanol/ antioxidant) 



Western blotting- Transfer (Blotting) 

Electrotransfer: 




Types: 

1. Wet transfer (gel and membrane fully 

immersed in transfer buffer) 

2. Semi-dry transfer (faster, consumes less 

buffer but less efficient!) 




Transfer buffers and running conditions: 




Membranes: 

1. Nitrocellulose 

membrane 

2. PVDF membrane 




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: 




Primary antibodies: 

Monoclonal: 

Less sensitive 

more specific 

Polyclonal: 

More sensitive 

less specific 




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 


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. 


Handbooks


Based on: 

2. Fluorescence 

(direct method using 

fluorophore labelled 

sec. Ab) 




Based on: 

3. Chemifluorescence 

(indirect method; 

ECF reaction) 


Principles and methods. 

28-9998-97. GE 


Western blotting- Imaging 

Types: 

1. Digital imaging: 

CCD camera-based 

imager or scanner 

CCD: charge-coupled device 

2. Chemiluminescence 

detection using X- 

ray film 

3. (Autoradiography) 

4. Colorimetric 

detection (HRP 

coupled sec Ab, 

peroxide and DAB) 

Western blotting. Principles and 

methods. 


Handbooks

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 


28-9998-97. GE Healthcare Handbooks 

Image analysis software is needed! (ImageQuant, Quantity One)

Western blotting- Analysis 

Example: 


Principles and methods. 

28-9998-97. GE 


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 


• Separation in capillaries filled 

with buffer solution: 


Sequencing of DNA

II. Capillary 


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


”Lab-on-a-Chip”: 

Rapid analysis of protein, DNA, and RNA 

in fluid samples (microfluidics) 

„lab-on-a-chip”


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


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!

Electrophoresis Western blotting

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