supported by: huro/0901/069/2.3.1 hu-ro-docs ... · aniko keller-pinter md phd luca mendler md phd...

ANIKO KELLER-PINTER MD PhD

LUCA MENDLER MD PhD

ElectrophoresisWestern blotting

Supported by:HURO/0901/069/2.3.1 HU-RO-DOCS

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 chargeb) applied voltage

Classification:I. Gel electrophoresis

- agarose or polyacrylamide gels - 1D (vertical / horizontal) or 2D - protein (native / urea / SDS) or DNA/RNA

II. Capillary electrophoresisIII. Microchip electrophoresis

I. Protein gel electrophoresis- general

migration

Large molecule, small charge

slow migration

Small molecule,high charge

fast migration

• agarose or polyacrylamide gels • 1D (vertical / horizontal) or 2D • protein (native / urea / SDS) or DNA

Separation

I. Protein gel electrophoresis- horizontal

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


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


I. Protein gel electrophoresis- vertical

Useful for: 1. Examining protein-protein interactions

2. Detecting protein isoforms

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


I. Protein gel electrophoresis- Native

• Separates denatured proteins by size and charge

• An useful technique to study protein modifications

migration


I. Protein gel electrophoresis- Urea

• 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

• 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

SDS: Sodium dodecyl sulfate

rod shaped protein

PAGE: Polyacrylamide gel electrophoresis

protein


I. Gel electrophoresis-SDS-PAGE

I. Protein gel electrophoresis-Two dimensional (2D)

• The first dimensionseparates 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

• agarose or polyacrylamide gels • 1D (vertical / horizontal) or 2D• protein (native / urea / SDS) or DNA

I. Protein gel electrophoresis-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)

• agarose or polyacrylamide gels • 1D (vertical / horizontal) or 2D• protein (native / urea / SDS) or DNA

I. Protein gel electrophoresis-Visualisation of proteins

• The position (heigth)of bands indicatestheir relative size

Coomassie blue dye Silver staining

Electrophoresis of serum proteins

• Agarose gel, native electrophoresis

Beta-1

Beta-2

Peaks are evaluated by densitometry

The figures are from http://www.sebia-usa.com/products/hyrys2.htmland http://erl.pathology.iupui.edu/LABMED/GENER27.HTM respectivelly (Feb 2007)

60% 3% 9% 12% 16%


Western blotting

Definition:

Western blotting. Principles and methods. 28-9998-97. GE Healthcare Handbooks

Western blotting- Sample preparation

• Use extraxtionmethods 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- 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- Transfer (Blotting)

Electro-transfer:



1. Wet transfer (gel and membrane fully immersed in transfer buffer)

2. Semi-dry transfer (faster, consumes less buffer but less efficient!)

Types:



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- Antibody probing

Blocking:



Primary antibodies: Polyclonal:More sensitiveless specific

Monoclonal:Less sensitivemore specific



Secondary antibodies:

• choice of enzyme-labeled antibodies: alkaline phosphatase (AP), horseradish peroxidase (HRP)

• biotinylated sec. Ab: three-layer system for low abundance targets

• 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!


Western blotting- Detection

Based on:1. Chemiluminescence

(indirect method; ECL reaction)



Based on:2. Fluorescence

(direct method usingfluorophore labelledsec. Ab)



Based on:3. Chemifluorescence

(indirect method; ECF reaction)


Western blotting- Imaging

Types:1. Digital imaging:

CCD camera-based imager or scanner

2. Chemiluminescencedetection using X-ray film

3. (Autoradiography)

4. Colorimetric detection (HRP coupled sec Ab, peroxide and DAB)

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

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


Western blotting- Analysis

Example:Western blotting. Principles and methods. 28-9998-97. GE Healthcare Handbooks

• 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)

I. Gel electrophoresis- DNA (RNA)

Visualization under UV-light after staining by ethidium bromide

• agarose or polyacrylamide gels

• 1D (vertical / horizontal) or 2D

• protein (native / urea / SDS) or DNA

II-III. Capillary and microchip electrophoresis

• rapid analysis

• automation

• low sample and reagent consumption

• high reproducibility due to standardization and automation

Advantages:

• Separation in capillaries filled with buffer solution:

Electrophoresis of serum proteins Sequencing of DNA

II. Capillary electrophoresis

DNA sequence electropherograms of the NOD2 gene.(Jane Alfred, Nature Reviews Genetics )


Sequencing of DNA

II. Capillary 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”

”Lab-on-a-Chip”: Rapid analysis of protein, DNA, and RNAin fluid samples (microfluidics)


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


• 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!


Advantages of microfluidics:

supported by: huro/0901/069/2.3.1 hu-ro-docs ... · aniko keller-pinter md phd luca mendler md phd...

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