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Two dimensional gel electrophoresis

Two dimensional gel electrophoresis. 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.

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Two dimensional gel electrophoresis

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  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 : 104 - 105 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

  11. Sample preparation B. Solubilise proteins: Lysis Solution Composition 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. NH3+ NH3+ NH2 COOH COO- COO- NH3+ NH3+ NH2 COOH COO- COO- pH < pI Net positive charge pH = pI pH > pI Net negative charge 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.

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

  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 SDS-PAGE Marker in paper 0.5% agarose in running buffer - + SDS-PAGE SDS-PAGE: Step 2 IPG strip in Equilibration buffer 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. Detection

  28. Detection Coommassie stain Silver stain

  29. Detection Copper stain SYPRO flourescent

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

  31. Analysis

  32. Analysis of spots • 2D gel software

  33. Analysis of spots • Commercial Software • Melanie 4 (GeneBio - Windows only) http://ca.expasy.org/melanie • ImageMaster 2D Elite (Amersham) http://www.imsupport.com/ • Phoretix 2D Advanced http://www.phoretix.com/ • PDQuest 6.1 (BioRad - Windows only) http://www.proteomeworks.bio-rad.com/html/pdquest.html

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

  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/ • http://www.abdn.ac.uk/~mmb023/protocol.htm • http://www.aber.ac.uk/~mpgwww/Proteome/Tut_2D.html • http://www.noble.org/PlantBio/MS/protocols.html

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