Electrophoresis Western blotting

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1 Supported by: HURO/0901/069/2.3.1 HU-RO-DOCS Electrophoresis Western blotting ANIKO KELLER-PINTER MD PhD LUCA MENDLER MD PhD

2 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

3 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

4 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

5 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

6 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

7 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

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

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

10 I. Protein gel electrophoresis- Two 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)

11 I. Protein gel electrophoresis- Visualisation of proteins The position (heigth) of bands indicates their relative size Coomassie blue dye Silver staining

12 Electrophoresis of serum proteins Agarose gel, native electrophoresis Beta-2 Beta-1

13 Electrophoresis of serum proteins Peaks are evaluated by densitometry 60% 3% 9% 12% 16% The figures are from and respectivelly (Feb 2007)

14 Western blotting Definition: Western blotting. Principles and methods GE Healthcare Handbooks

15 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 GE Healthcare Handbooks

16 Western blotting- Gel electrophoresis Western blotting. Principles and methods GE Healthcare Handbooks

17 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. Principles and methods GE Healthcare Handbooks

18 Western blotting- Transfer (Blotting) Electrotransfer: Western blotting. Principles and methods GE Healthcare Handbooks

19 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 GE Healthcare Handbooks

20 Western blotting- Transfer (Blotting) Transfer buffers and running conditions: Western blotting. Principles and methods GE Healthcare Handbooks

21 Western blotting- Transfer (Blotting) Membranes: 1. Nitrocellulose membrane 2. PVDF membrane Western blotting. Principles and methods GE Healthcare Handbooks

22 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 GE Healthcare Handbooks

23 Western blotting- Antibody probing Blocking: Western blotting. Principles and methods GE Healthcare Handbooks

24 Western blotting- Antibody probing Primary antibodies: Monoclonal: Less sensitive more specific Polyclonal: More sensitive less specific Western blotting. Principles and methods GE Healthcare Handbooks

25 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: 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 GE Healthcare Handbooks

26 Western blotting- Detection Based on: 1. Chemiluminescence (indirect method; ECL reaction) Western blotting. Principles and methods GE Healthcare Handbooks

27 Western blotting- Detection Based on: 2. Fluorescence (direct method using fluorophore labelled sec. Ab) Western blotting. Principles and methods GE Healthcare Handbooks

28 Western blotting- Detection Based on: 3. Chemifluorescence (indirect method; ECF reaction) Western blotting. Principles and methods GE Healthcare Handbooks

29 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 GE Healthcare Handbooks

30 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 GE Healthcare Handbooks Image analysis software is needed! (ImageQuant, Quantity One)

31 Western blotting- Analysis Example: Western blotting. Principles and methods GE Healthcare Handbooks

32 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)

33 II-III. Capillary and microchip electrophoresis Advantages: rapid analysis automation low sample and reagent consumption high reproducibility due to standardization and automation

34 II. Capillary electrophoresis Separation in capillaries filled with buffer solution: Electrophoresis of serum proteins Sequencing of DNA

35 II. Capillary electrophoresis Sequencing of DNA DNA sequence electropherograms of the NOD2 gene. (Jane Alfred, Nature Reviews Genetics ) Electrophoresis of serum proteins

36 III. Microchip electrophoresis microchip tiny channels manufactured in glass or plasctic that serve as pathways for the movement of fluid samples

37 III. Microchip electrophoresis Lab-on-a-Chip : Rapid analysis of protein, DNA, and RNA in fluid samples (microfluidics) lab-on-a-chip

38 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

39 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!