1. Two dimensional gel electrophoresis

2. What is 2D gel electrophoresis?
Separation and identification of proteins in a sample by displacement in 2 dimensions oriented at right-angle to one another.
First dimension: Isoelectric focussing
Second dimension: SDS PAGE

3. Overview of 2D gel electrophoresis

4. Basis of 2D gel electrophoresis
Highly unlikely for proteins to be similar in two distinct properties
Isoelectric point
Protein mass in SDS PAGE
Protein complex mass in native PAGE

5. Advantages of 2D gel electrophoresis
Resolution : proteins from a sample.
Preset conditions ( pH ranges, size of the gel, staining methods etc) can be changed to increase resolution.
Delivers a map of intact protein that can be stored and analyzed at will.
One of the core technology of proteomics.

6. Limitations Hard to resolve very acidic or very basic proteins
very low or very high molecular weight.
Poor resolution
hydrophobic or membrane bound
nuclear proteins
Sample size
proteins cannot be amplified like DNA.
Difficulty in process automation

7. 2D gel electrophoresis steps
Sample preparation
First dimension separation
Isoelectric focussing
Second dimension separation
SDS PAGE
Detection of spots
Analysis of protein spots

8. Sample preparation

9. Sample preparation
Must remove substances that might interfere with separation process such as salts, polar detergents (SDS), lipids, polysaccharides, nucleic acids
Must try to keep proteins soluble during both phases of electrophoresis process

10. Sample preparation A. Contaminants and their removal procedure
Particle
Effect
Removal
Charged molecules
E.g.: Salts, buffer components & nucleotides
Poor focussing
Dialysis or gel filtration
SDS
Proteins move towards positive electrode
Acetone precipitation
DNA
Clog the gel pores
Interfere with staining
DNase or sonication
Particulate matter
Centrifugation

11. Sample preparation B. Solubilise proteins: Lysis Solution Composition
Component
Function
Final Concn
Urea
(fresh & deionised)
Solubilises proteins
8M-9M
Detergents:
Non-ionic/ zwitter ionic
(CHAPS, CHAPSO, NP-40)
Maintains proteins in solution during rehydration
0.5-4%
Reducing Agent: DTT/ DTE
Cleaves sulfide bonds
20-100mM
Ampholytes (3-10)
Maintaining the pH gradient
0.2-2%
CHAPS: 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate
DTT: Dithrioerythritol; DTE: epimer of DTT

12. Sample preparation: Protocol

13. Isoelectric focussing

14. Isoelectric focussing
Separation on basis of pI, not Mw
Requires very high voltages (5000V)
Requires a long period of time (10h)
Presence of a pH gradient is critical
Uses ampholytes to establish pH gradient

15. Isoelectric focussing
Isoelectric point is the pH of a solution at which the net charge of protein is zero. In electrophoresis there is no motion of the particles in an electric field at the isoelectric point.
NH3+
COOH
pH < pI
Net positive charge
COO-
pH = pI
NH2
pH > pI
Net negative charge

16. Isoelectric focussing
pH gradients
Soluble Ampholytes
heterogeneous polymers of charged compounds in a regular (tube) polyacrylamide gel
Variability: batch to batch
pH range about 4-8 at equilibrium
Immobilised pH gradient
Acrylamide derivatives with charged side chains immobilised on a strip
Stable and reproducible gradient
Higher mechanical strength
Higher protein loading capacity

17. Isoelectric focussing
IPG strips (3 mm x 18 cm x 0.5 mm)
Narrow range
Medium range
Broad range 4 7
3.5
4.5
5.5
6.7
4.0
5.0
6.0 3 10 6 11

18. Isoelectric focussing
Load sample per groove
Peel off protective film from strip
Place IPG strip gel facing down in groove
Rehydrate overnight (~22 hrs) at room temperature

19. Isoelectric focussing
IEF run
Slowly increase the voltage and apply a high voltage at end to obtain sharp narrow zones of protein

20. Isoelectric focussing
Equilibration
Reduction :
50mM DTT in equilibration buffer for 15 minutes
Maintains proteins in a reduced state
Alkylation (optional):
125mM iodoacetamide in equilibration buffer for 15 minutes
Alkylating sulfhydryl group reduces vertical streaking
Equilibration buffer: 6M urea, 30% glycerol; 1.6% SDS; 0.002% bromophenol blue; 45 mM Tris base (pH 7.0)

21. SDS PAGE

22. SDS-PAGE Second dimension SDS-PAGE steps
Equilibrating IPG strips after IEF.
Applying IPG strips to the second dimension SDS gel.
Performing SDS-PAGE

23. SDS-PAGE: Step1 After IEF run Remove the IPG strip from the tray
Place IPG strip facing up in the equilibration buffer + -

24. SDS-PAGE: Step 2 IPG strip in Equilibration buffer+ -
IPG strip in Equilibration buffer
SDS-PAGE
0.5% agarose
in running buffer
Marker in paper
IPG strip is placed on top of pre-cast SDS-PAGE gel and electric current applied

25. SDS-PAGE: Step 3 Separation on basis of MW, not pI
Requires modest voltages (200V)
Requires a shorter period of time (2h)
Presence of SDS is critical to disrupting structure and making mobility
Degree of resolution determined by %acrylamide & electric field strength

26. Detection

27. Sensitivity (ng/spot)
Detection
Stain
Sensitivity (ng/spot)
Advantages
Coomassie R-250
50-100
Simple, fast, consistent
Silver stain
1-4
Very sensitive, awkward
Copper stain
5-15
Reversible, simple, fast
Zinc stain
SYPRO ruby
1-10
Very sensitive, fluoroscent
Western blotting
Directly used for sequencing

28. Coommassie stain Silver stain
Detection
Coommassie stain Silver stain

29. Copper stain SYPRO flourescent
Detection
Copper stain SYPRO flourescent

30. Insufficient equilibration, insufficient SDS
Trouble shooting
Horizontal streaks
Sample not completely solubilized prior to application on IPG, sample poorly soluble in rehydration solution, ionic impurities, ionic detergents
Vertical streaks
Insufficient equilibration, insufficient SDS

31. Analysis

32. Analysis of spots
2D gel software

33. Analysis of spots Commercial Software
Melanie 4 (GeneBio - Windows only)
ImageMaster 2D Elite (Amersham)
Phoretix 2D Advanced
PDQuest 6.1 (BioRad - Windows only)

34. Analysis of spots Common Software Features Image contrast and coloring
Gel annotation (spot selection & marking)
Automated peak picking
Spot area determination (Integration)
Matching 2 gels
Stacking/Aligning/Comparing gels

35. Analysis Excision the spots of interest
Pick up the protein gel spot from gel
Manual
Automatic
In-gel digestion:
Washing process
Dehydratation and drying
Trypsin digestion (50 ng trypsin, 37C 16h)
Extraction
Desalt and concentrate the peptide

36. Analysis Identification of eluted protein spots MALDI-TOF
(Matrix Assisted Laser Desorption/Ionization-Time Of Flight)
MS (Mass Spectrometry),
Peptide mass fingerprint

37. Applications Analyzing proteome profiles
Detecting post- or co-translational modifications
Discovering new drug targets
Studying protein expression in normal, disease, or developmental states
Identifying novel proteins

38. (?????) Normal cells Tumor SDS-PAGE isoelectrofocusing
Laser-captured microdissector (LCM)
(?????)
Clinical specimens
Cryostat
2D gel
electrophoresis
Immage system

39. Recap

40. Schedule of a 2D Experiment
Day 1: Sample preparation and IEF
1. Load protein sample onto IPG strip (IEF)
2. Run the IEF (about 24 hours)
3. Polyacrylamide gel casting
Day 2: Equilibrium IPG strip and running SDS-PAGE
1. Remove IPG strip from IEF machine
2. Equilibrium IPG strip
3. Put IPG strip onto SDS-PAGE
4. Run the SDS-PAGE (overnight)

41. Schedule of a 2D Experiment
Day 3: Staining, image scanning and image analysis
1. Remove the gel from the cassette
2. Stain the gel by SYPRO Ruby or silver
3. Scan the gel image
4. Image analysis
Day 4: In-gel digestion, MALDI-TOF and database search
1. Pick the protein gel spot from gel
2. In-gel digestion
3. Spot the sample onto MALDI chip
4. MALDI-TOF analysis
5. Database search

42. Database of 2D maps

43. Online protocols http://ca.expasy.org/ch2d/protocols/