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2D Gel Analysis

Lecture 1.3. 2. Separation

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2D Gel Analysis

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    1. Lecture 1.3 1 2D Gel Analysis David Wishart University of Alberta Edmonton, AB david.wishart@ualberta.ca

    2. Lecture 1.3 2 Separation & Display Tools 1D Slab Gel Electrophoresis 2D Gel Electrophoresis Capillary Electrophoresis HPLC (SEC, IEC, RP, Affinity, etc.) Protein Chips

    3. Lecture 1.3 3 2D Gel Electrophoresis Simultaneous separation and detection of ~2000 proteins on a 20x25 cm gel Up to 10,000 proteins can be seen using optimized protocols

    4. Lecture 1.3 4 Why 2D GE? Oldest method for large scale protein separation (since 1975) Still most popular method for protein display and quantification Permits simultaneous detection, display, purification, identification, quantification Robust, increasingly reproducible, simple, cost effective, scalable & parallelizable Provides pI, MW, quantity

    5. Lecture 1.3 5 Steps in 2D GE Sample preparation Isoelectric focusing (first dimension) SDS-PAGE (second dimension) Visualization of proteins spots Identification of protein spots Spot pattern evaluation/annotation

    6. Lecture 1.3 6 Steps in 2D GE

    7. Lecture 1.3 7 Sample Preparation Sample preparation is key to successful 2D gel experiments Must break all non-covelent protein-protein, protein-DNA, protein-lipid interactions, disrupt S-S bonds Must prevent proteolysis, accidental phosphorylation, oxidation, cleavage, deamidation

    8. Lecture 1.3 8 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

    9. Lecture 1.3 9 Cell Disruption Methods Sonication French press Glass bead disruption Enzymatic lysis Detergent lysis Freeze-thaw Osmotic lysis

    10. Lecture 1.3 10 Sample Preparation PMSF Pefabloc EDTA EGTA leupeptin Dialysis Filtration Centrifugation Chromatography Solvent Extraction

    11. Lecture 1.3 11 Protein Solubilization 8 M Urea (neutral chaotrope) 4% CHAPS (zwitterionic detergent) 2-20 mM Tris base (for buffering) 5-20 mM DTT (to reduce disulfides Carrier ampholytes or IPG buffer (up to 2% v/v) to enhance protein solubility and reduce charge-charge interactions

    12. Lecture 1.3 12 Other Considerations Further purification or separation? Subcellular fractionation Chromatographic separation Affinity purification Optimizing electrophoresis parameters IEF pH gradient, Acrylamide %, loading Limits of detection ng? (Coomasie stain) pg or fg? (Western)

    13. Lecture 1.3 13 Detergent Fractionation

    14. Lecture 1.3 14 Subcellular Fractionation

    15. Lecture 1.3 15 Differential Solubilization

    16. Lecture 1.3 16 Steps in 2D GE Sample preparation Isoelectric focusing (first dimension) SDS-PAGE (second dimension) Visualization of proteins spots Identification of protein spots Spot pattern evaluation/annotation

    17. Lecture 1.3 17 2D Gel Principles

    18. Lecture 1.3 18 Isoelectric Focusing (IEF)

    19. Lecture 1.3 19 IEF Principles

    20. Lecture 1.3 20 Isoelectric Focusing Separation of basis of pI, not Mw Requires very high voltages (5000V) Requires a long period of time (10h) Presence of a pH gradient is critical Degree of resolution determined by slope of pH gradient and electric field strength Uses ampholytes to establish pH gradient

    21. Lecture 1.3 21 Ampholytes vs. IPG Ampholytes are small, soluble, organic molecules with high buffering capacity near their pI (not characterized) Used to create pH gradients via user Gradients not stable Batch-to-batch variation is problematic An immobilized pH gradient (IPG) is made by covalently integrating acrylamido buffer molecules into acrylamide matrix at time of gel casting Stable gradients Pre-made (at factory) Simplified handling

    22. Lecture 1.3 22 IPG Strips

    23. Lecture 1.3 23 Narrow-Range IPG Strips

    24. Lecture 1.3 24 IEF Phase of 2D GE

    25. Lecture 1.3 25 Steps in 2D GE Sample preparation Isoelectric focusing (first dimension) SDS-PAGE (second dimension) Visualization of proteins spots Identification of protein spots Spot pattern evaluation/annotation

    26. Lecture 1.3 26 SDS PAGE

    27. Lecture 1.3 27 SDS PAGE Tools

    28. Lecture 1.3 28 SDS PAGE Principles

    29. Lecture 1.3 29 SDS-PAGE Principles

    30. Lecture 1.3 30 Mobility & Acrylamide%

    31. Lecture 1.3 31 Electrophoretic Mobility

    32. Lecture 1.3 32 SDS-PAGE Separation of 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 ~ 1/MW Degree of resolution determined by %acrylamide & electric field strength

    33. Lecture 1.3 33 SDS-PAGE for 2D GE After IEF, the IPG strip is soaked in an equilibration buffer (50 mM Tris, pH 8.8, 2% SDS, 6M Urea, 30% glycerol, DTT, tracking dye) IPG strip is then placed on top of pre-cast SDS-PAGE gel and electric current applied This is equivalent to pipetting samples into SDS-PAGE wells (an infinite #)

    34. Lecture 1.3 34 SDS-PAGE for 2D GE

    35. Lecture 1.3 35 2D Gel Reproducibility

    36. Lecture 1.3 36 Trouble Shooting 2D GE

    37. Lecture 1.3 37 Advantages and Disadvantages of 2D GE Provides a hard-copy record of separation Allows facile quantitation Separation of up to 9000 different proteins Highly reproducible Gives info on Mw, pI and post-trans modifications Inexpensive Limited pI range (4-8) Proteins >150 kD not seen in 2D gels Difficult to see membrane proteins (>30% of all proteins) Only detects high abundance proteins (top 30% typically) Time consuming

    38. Lecture 1.3 38 2D Gel Protocols & Courses 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 1 Day and 1 Week Courses http://us.expasy.org/bprg/training/(Geneva) http://www.pence.ca (Toronto)

    39. Lecture 1.3 39 Steps in 2D GE Sample preparation Isoelectric focusing (first dimension) SDS-PAGE (second dimension) Visualization of proteins spots Identification of protein spots Spot pattern evaluation/annotation

    40. Lecture 1.3 40 Protein Detection Coomassie Stain (100 ng to 10 mg protein) Silver Stain (1 ng to 1 mg protein) Fluorescent (Sypro Ruby) Stain (1 ng & up)

    41. Lecture 1.3 41 Gel Stains - Summary

    42. Lecture 1.3 42 Stain Examples

    43. Lecture 1.3 43 Stain Examples

    44. Lecture 1.3 44 Detection via Western Blot

    45. Lecture 1.3 45 Imaging/Scanning Tools

    46. Lecture 1.3 46 Steps in 2D GE Sample preparation Isoelectric focusing (first dimension) SDS-PAGE (second dimension) Visualization of proteins spots Identification of protein spots Spot pattern evaluation/annotation

    47. Lecture 1.3 47 2D-GE + MALDI (PMF)

    48. Lecture 1.3 48 2D-GE + MS-MS

    49. Lecture 1.3 49 Typical Results 401 spots identified 279 gene products Confirmed by SAGE, Northern or Southern Confirmed by amino acid composition Confirmed by amino acid sequencing Confirmed by Mw & pI

    50. Lecture 1.3 50 Steps in 2D GE Sample preparation Isoelectric focusing (first dimension) SDS-PAGE (second dimension) Visualization of proteins spots Identification of protein spots Spot pattern evaluation/annotation

    51. Lecture 1.3 51 2D Gel Software

    52. Lecture 1.3 52 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

    53. Lecture 1.3 53 Common Software Features Image contrast and coloring Gel annotation (spot selection & marking) Automated peak picking Spot area determination (Integration) Matching/Morphing/Landmarking 2 gels Stacking/Aligning/Comparing gels Annotation copying between 2 gels

    54. Lecture 1.3 54 2D Gel Analysis Freeware

    55. Lecture 1.3 55 2D Gel Analysis Freeware

    56. Lecture 1.3 56 Flicker Permits comparison of 2 images from internet sources on web browser Comparison via adjustable flicker rate of overlaid gel images Images may be enhanced by spatial warping, 3D projections or relief map, image sharpening, contrast enhancement, zooming, complement grayscale transform

    57. Lecture 1.3 57 2D Gel Analysis Freeware

    58. Lecture 1.3 58 2D Gel Analysis Freeware

    59. Lecture 1.3 59 Federated 2D Gel Databases Remotely queryable via the web Attainable through SWISS-PROT search Linked to other 2D databases via web Image mapped 2D gel spots to support graphical image query Directly reachable from within 2D gel analysis software

    60. Lecture 1.3 60 2D Gel Databases

    61. Lecture 1.3 61 2D Gel Databases

    62. Lecture 1.3 62 Swiss 2D-PAGE

    63. Lecture 1.3 63 Swiss 2D-PAGE

    64. Lecture 1.3 64 Swiss 2D-PAGE

    65. Lecture 1.3 65 Competing Technologies

    66. Lecture 1.3 66 ICAT vs 2D Gels

    67. Lecture 1.3 67 MudPIT

    68. Lecture 1.3 68 2D Gels vs Protein Arrays

    69. Lecture 1.3 69 A Triumph For Gels (Actually Western Blotting)

    70. Lecture 1.3 70 Yeast Proteome Analysis

    71. Lecture 1.3 71 Tap Tagged Western

    72. Lecture 1.3 72 Tap-Tagged Western - Sensitivity

    73. Lecture 1.3 73 Yeast Proteome Results

    74. Lecture 1.3 74 The Yeast Proteome 80% of the proteome is expressed during normal growth conditions Abundance of proteins ranges from fewer than 50 to more than 106 molecules per cell Many proteins, including essential proteins and most transcription factors, are present at levels that are not readily detectable by other proteomic techniques

    75. Lecture 1.3 75 Conclusions 2D gel electrophoresis is still the most popular and powerful method for protein display, separation, visualization and quantitation Offers good to excellent sensitivity and is now very reproducible 2D GE is still essential for proteomics Running and analyzing 2D gels requires skill, patience and good software

    76. Lecture 1.3 76 Conclusions Web tools are now available that permit partial analysis and comparison of 2D gels Commercial software still is required in most cases to complete full-scale analysis Web-enabled gel databases are now democratizing & popularizing 2D gel analysis Competing technologies are now emerging that may offer advantages over 2DE

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