2. Purification of Green Fluorescent Protein (GFP)

3. 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 Instructors

4. Why Teach Protein Purification? Powerful teaching tool Laboratory extensions Real-world connections Link to careers and industry Standards based

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

7. Workshop Time Line Introduction Purify GFP using column chromatography Overview of how to separate GFP via SDS- PAGE

8. Central Framework of Molecular Biology DNA RNA ProteinTrait

9. 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

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

12. Gene Expression Beta Lactamase –Ampicillin resistance Green Fluorescent Protein (GFP) –Aequorea victoria jellyfish gene araC regulator protein –Regulates GFP transcription

13. Transcriptional Regulation Lactose operon Arabinose operon pGLO plasmid

14. Transcriptional Regulation BAD araC BAD RNA Polymerase Effector (Arabinose) araC BAD ara Operon RNA Polymerase ZYA ZYA LacI Effector (Lactose) ZYA LacI lac Operon

15. Gene Regulation RNA Polymerase araC ara GFP Operon GFP Gene araC GFP Gene araC GFP Gene Effector (Arabinose) BAD araC BAD RNA Polymerase Effector (Arabinose) araC BAD ara Operon

16. GFP Chromatography Kit

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

18. Volume Measurement

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

20. GFP Animation

21. Column Chromatography Chromatography used for protein purification –Size exclusion –Ion exchange –Hydrophobic interaction

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

23. 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 H 2 O Hydrophobic bead N H H H + H - + + O S O O O - - O S O O O - - N H H H + H - + + O S O O O - - O S O O O - -

24. 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 N H H H + H - + + O S O O O - - O S O O O - - Hydrophobic bead - + + - + + - + +

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

26. Laboratory Quick Guide

27. 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

28. 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/ampLB/amp/ara

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

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