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Purification of Green Fluorescent Protein (GFP)

Purification of Green Fluorescent Protein (GFP). Instructors. Stan Hitomi Coordinator – Math & Science Principal – Alamo School San Ramon Valley Unified School District Danville, CA Kirk Brown Lead Instructor, Edward Teller Education Center Science Chair, Tracy High School

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Purification of Green Fluorescent Protein (GFP)

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  1. Purification of Green Fluorescent Protein (GFP)

  2. Instructors Stan Hitomi Coordinator – Math & Science Principal – Alamo School San Ramon Valley Unified School District Danville, CA Kirk Brown Lead Instructor, Edward Teller Education Center Science Chair, Tracy High School and Delta College, Tracy, CA Bio-Rad Curriculum and Training Specialists: Sherri Andrews, Ph.D. sherri_andrews@bio-rad.com Essy Levy, M.Sc. essy_levy@bio-rad.com Leigh Brown, M.A. leigh_brown@bio-rad.com

  3. WhyTeachProtein Purification? • Powerful teaching tool • Laboratory extensions • Real-world connections • Link to careers and industry • Standards based

  4. Green Fluorescent Protein (GFP) Chromatography Kit GFP Purification Kit Advantages • Cloning in action • Links to biomanufacturing • Biopharmaceutical development • Amazing visual results

  5. WorkshopTime Line • Introduction • Purify GFP using column chromatography • Overview of how to separate GFP via SDS-PAGE

  6. DNA RNA Protein Trait Central Framework of Molecular Biology

  7. Links to Real-world • GFP is a visual marker • Study of biological processes (example: synthesis of proteins) • Localization and regulation of gene expression • Cell movement • Cell fate during development • Formation of different organs • Screenable marker to identify transgenic organisms

  8. Using GFP as a biological tracer http://www.conncoll.edu/ccacad/zimmer/GFP-ww/prasher.html With permission from Marc Zimmer

  9. GeneExpression • Beta Lactamase • Ampicillin resistance • Green Fluorescent Protein (GFP) • Aequorea victoria jellyfish gene • araC regulator protein • Regulates GFP transcription

  10. Transcriptional Regulation • Lactose operon • Arabinose operon • pGLO plasmid

  11. ara Operon lac Operon araC B A D LacI Z Y A Effector (Arabinose) Effector(Lactose) araC B A D LacI Z Y A RNA Polymerase RNA Polymerase B A D araC Z Y A Transcriptional Regulation

  12. ara GFP Operon ara Operon araC GFP Gene araC B A D Effector(Arabinose) Effector (Arabinose) araC B A D araC GFP Gene RNA Polymerase RNA Polymerase B A D araC araC GFP Gene Gene Regulation

  13. GFP Chromatography Kit

  14. GFP Purification Procedures Overview Day 2 Day 1 Day 3

  15. Volume Measurement

  16. Why Use Chromatography? • To purify a single recombinant protein of interest from over 4,000 naturally occurring E. coli gene products.

  17. GFP Animation

  18. Column Chromatography • Chromatography used for protein purification • Size exclusion • Ion exchange • Hydrophobic interaction

  19. Hydrophobic Interaction Chromatography:(HIC)Steps 1–3 • Add bacterial lysate to column matrix in high salt buffer 2. Wash less hydrophobic proteins from column in low salt buffer • Elute GFP from column with no salt buffer

  20. Hydrophobic bead H H O O - - - - H H + + O O O O S S N N H H H H O O + + O O - - - - O O S S O O - - O O + + Step 1:Hydrophobic Interaction Chromatography • Add bacterial lysate to column matrix in high salt buffer • Hydrophobic proteins interact with column • Salt ions interact with the less hydrophobic proteins and H2O

  21. Hydrophobic bead H O - - + H O O S N H H O + + + + O - - - - - O S O - O + + + + Step 2:Hydrophobic Interaction Chromatography • Wash less hydrophobic from column with low salt buffer • Less hydrophobic E. coli proteins fall from column • GFP remains bound to the column

  22. + + + + + - - - - - + + + + + Step 3:Hydrophobic Interaction Chromatography Hydrophobic bead • Elute GFP from column by adding a no-salt buffer GFP • Released from column matrix • Flows through the column

  23. LaboratoryQuick Guide

  24. Helpful Hints:Hydrophobic Interaction Chromatography • Add a small piece of paper to collection tube where column seats to insure column flow • Rest pipet tip on side of column to avoid column bed disturbance when adding solutions • Drain until the meniscus is just above the matrix for best separation

  25. GFP Electrophoresis Extension • SDS PAGE sample preps are made from white and green colonies • Bacterial lysates are prepared in Laemmli buffer • Samples are loaded onto polyacrylamide gels LB/amp LB/amp/ara

  26. GFP Visualization-During & Post Electrophoresis M W G M W G M W G • Samples are electrophoresed • Fluorescent GFP can be visualized during electrophoresis • Coomassie stained gels allow for visualization of induced GFP proteins Fluorescent isoform Non-fluorescent isoform During Electrophoresis Post Electrophoresis Prestained bands + UV activated GFP Fluorescent bands Coomassie stained bands

  27. Webinars • Enzyme Kinetics — A Biofuels Case Study • Real-Time PCR — What You Need To Know and Why You Should Teach It! • Proteins — Where DNA Takes on Form and Function • From plants to sequence: a six week college biology lab course • From singleplex to multiplex: making the most out of your realtime experiments explorer.bio-rad.comSupportWebinars

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