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Protein Analysis Workshop with Two-Dimensional Gel Electrophoresis

Explore the world of proteomics with Dr. Yu Liang at the Proteomics Core Facility. From genotype to phenotype, delve into the detection of post-translational modifications through spot detection, image analysis, and protein identification using peptide sequencing via mass spectrometry database search. Benefit from customized core services, including new phosphoprotein staining and difference gel electrophoresis. Get hands-on experience with cutting-edge equipment and comprehensive 2-D gel services. Visit www.med.uc.edu/proteomics for more details.

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Protein Analysis Workshop with Two-Dimensional Gel Electrophoresis

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  1. CEG Protein Analysis Workshop Two-Dimensional Gel Electrophoresis Yu Liang, Ph.D. Proteomics Core Facility at UC Medical Center

  2. From Genotype to Phenotype • Genome: DNAs • Transcriptome: RNAs • Proteome: Proteins • Physiome: Metabolites • Biome: Environment

  3. Beyond the Genome • Proteins are ultimately responsible for all biological processes that take place within cells. • Protein dynamics reflect the state of biological system at a given time • Detection and identification of post-translational modifications (PTM)

  4. Proteomics Overview Spot Detection & Image Analysis Sample Preparation 2-D Electrophoresis (I) (II) (III) Enzymatic Digestion Peptide-Mass Fingerprinting Protein Identification Peptide Sequencing via MS Database Search (IV) (V) (VI)

  5. Personnel • Yu Liang, Ph.D. Research Associate MSB 5301 Tel: 558-2347 liangyu@ucmail.uc.edu http://www.med.uc.edu/proteomics/ • John Maggio, Ph.D. Professor and Chair Department of Pharmacology & Cell Biophysics

  6. Customized Core Services • Complete 2-D Gel Service with Spot Picking for MS Analysis • Complete 2-D Gel Service, or (1) IEF Only (2) SDS-PAGE (large format) Only (3) SDS-PAGE Plus (staining & imaging) (4) Gel Staining (fluorescent, Sypro Ruby) (5) Image Analysis Only (6) Image Analysis Plus (spot pick) • Two new services: (1) Phosphoprotein staining by Pro-Q Diamond fluorescent dye (2) Difference gel electrophoresis (DIGE) by Cydye labelling

  7. Proteomics Core Equipment • Genomic Solutions Investigator 2-D Electrophoresis System • Genomic Solutions Gel Casting System • Genomic Solutions ProImage Image Acquisition System • Dell Optiplex Image Analysis Computers • New: • Fuji Fluorescent Image Analyzer FLA5100 (Phosphoprotein staining and DIGE)

  8. Genomic Solutions 2D Electrophoresis System pH phaser isoelectric focusing system Investigator 2-D electrophoresis running system

  9. pH low High MW High low 2-D Image (Sypro Ruby) • mouse cardiac • 250 g loading • pH 3-10 IEF strips • 12.5% SDS-PAGE • File ID: mb699

  10. Sample gels Control Experimental

  11. 2-D Image Control Experimental • mouse cardiac; 250 g loading; pH 3-10 IEF strips; 12.5% SDS-PAGE; file ID: sc13con vs. sc08iso; spot ID: S8-1

  12. 2-D Image Experimental Control • mouse cardiac; 250 g loading; pH 3-10 IEF strips; 12.5% SDS-PAGE; file ID: sc13con vs. sc08iso

  13. PTM? Downregulation? 2-D Image Control Experimental • mouse cardiac; 250 g loading; pH 3-10 IEF strips; 12.5% SDS-PAGE; file ID: sc5bcon vs. sc15iso

  14. 2-D Gel Analysis

  15. 2-D Gel Analysis

  16. 2-D Gel Analysis

  17. Deliverables • Fluorescence-stained 2-D gels • Electronic image files • Spot list • Normalized volumes of spots Plus • Free access to our software for further analysis • Free advice and consultation on further protein characterization as well as 2-D image analysis

  18. New Website

  19. www.med.uc.edu/proteomics or proteomics.uc.edu • Contact • Protocols • Price list • Services • News & Events • Feedback • Publications • On-line order • FAQs

  20. FAQs • How do I get started? • What do I get for my result? • How much protein should I load for the 2-D gel analysis? • How long will it take to get the results? • Who is responsible for preparation of protein samples? • What protocol should I use for protein solubilization?

  21. Sample Preparation • The most important step towards the success of 2-D electrophoresis • Basic rules: keep everything simple keep samples cool use high-purity reagents • Keep proteins denatured and in solution: 7M urea, 2M thiourea, and 4% CHAPS. • Break disulfide bonds: 20 mM DTT. (NOT 2-ME) • Prevent protein modification: protease inhibitors; phosphatase inhibitors. • NO ionic detergent, esp. SDS. Non-ionic detergents are OK, e.g. Triton X-100 and NP-40. • Keep salt concentration below 10 mM. • Clean up interfering substance, including salt, nucleic acids, lipids, and polysaccharides: acetone (TCA) precipitation and commercial kits. • Protein quantity measurement

  22. New Fuji FLA 5100 • Pro-Q diamond fluorescent staining for phosphoproteins • Multiplexing using DIGE

  23. Multiplexing by Difference Gel Electrophoresis (DIGE) DIGE with CyDye labeling of protein (Amersham Web Site)

  24. Multiplexing by Difference Gel Electrophoresis (DIGE) • Comparison of samples within the same geleliminates gel-to-gel variation • Including the internal standards improves statistical confidence of comparing samples for protein abundance changes • Reduces number of gels needed to be run in the experiment: 2 comparing groups with 6 individuals, 36 gels for regular 2-D gels 12 gels for 2-dye DIGE 6 gels for 3-dye DIGE

  25. Post-Translational Modifications • Important for biological processes, particularly signal transductions • Most cellular processes are regulated by reversible phosphorylation of proteins • Studies of phosphorylated proteins involve radioisotope-labeling and specific antibodies

  26. Pro-Q Diamond Staining of a 2-D gel • Selectively stains phosphoproteins in 2-D gel • Allows direct in-gel detection of phosphate groups attached to tyrosine, serine, or threonine residues • NO NEED for antibody and radioisotope • Signal correlates with the number of phosphate groups • Fully compatible with mass spectrometry • Ratio of Pro-Q diamond to Sypro Ruby =phosphorylation level/total protein

  27. Pro-Q Diamond staining Blue: Pro-Q Diamond phosphoprotein gel stain Red: SYPRO Ruby total protein stain (Molecular Probes Website)

  28. Samples Wanted… • Among 0.5~1 million proteins in human proteome, what’s your favorite one? We are here to help the hunting. • Please send your samples to: Proteomics Core MSB5301 Tel: (513) 558-2347

  29. Simon Chu – Pharmacology Yolanda Sanchez – Mol. Genetics Keith Jones – Pharmacology Kathleen Dixon – Env. Health Leslie Myatt – OB/GYN Ron Millard – Pharmacology Alex Lentsch – Surgery William Martin – Dean’s Lab/Administrative Evangelia Kranias – Pharmacology Jeff Molkentin – CHMC/Mol. Card. Biology Jeff Robbins – CHMC/Mol. Card. Biology Jennifer Maller – CHMC/Plastic Surgery Alvaro Puga – Environmental Health Howard Shertzer – Envoironmental Health Frank Sharp – Neurology Erik Knudsen – Cell Biology Karen Knudsen – Cell Biology Raymond Boissy – Dermatology Jodie Duffy – CHMC/Cardiovascular Surgery Kristen Page – CHMC/Critical Care Joanna Groden – Molecular Genetics David Wieczorek – Molecular Genetics Joan Cook-Mills – Pathology Muhammed Ashraf – Pathology Jim Stringer – Molecular Genetics Ralph Giannella – IM/Digestive Disease Steven Keller – Biology Joseph Solomkin – Surgery Tom Shanley – CHMC/Critical Care Steven Chernausek – CHMC/Endocrinology George Leikauf – Environmental Health Michael Borchers – Environmental Health Gary Shull – Molecular Genetics Jeff Matthews – Surgery Mary-Beth Genter – Environmental Health Current/Prospective Clients

  30. Special Thanks… • Dr. John Maggio • Dr. Limbach’s MS Core Dr. Stephen Macha http://www.chembus.uc.edu/massnew/maintbl.asp

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