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Developing a Fluorescamine Assay to Probe Cardiac Protein Structure

Developing a Fluorescamine Assay to Probe Cardiac Protein Structure. Virginia Dines and Siddharth Damania 2006-2007. Thomas Biochemistry Lab University of Minnesota. Introduction. Nearly 60 million Americans suffer from heart disease

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Developing a Fluorescamine Assay to Probe Cardiac Protein Structure

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  1. Developing a Fluorescamine Assay to Probe Cardiac Protein Structure Virginia Dines and Siddharth Damania 2006-2007

  2. Thomas Biochemistry Lab University of Minnesota

  3. Introduction • Nearly 60 million Americans suffer from heart disease • Cardiac muscles require calcium to flow through the membrane calcium pump to beat Used with Permission from Dr. Thomas

  4. Phospholamban Robia, Flohr & Thomas (2005), Biochemistry 44: 4302 – 4311 Oxenoid & Chou (2005), PNAS 102: 10870 – 10875

  5. Phospholamban

  6. Phospholamban

  7. Spin Label

  8. Spin Label

  9. Phospholamban

  10. Phospholamban

  11. Phospholamban

  12. Fluorescamine

  13. Fluorescamine

  14. Fluorescamine

  15. Background • Study by Udenfriend et al. (1972) showed that when fluorescamine is reacted with primary amines (in lysine and PLB), the fluorescent intensity of the fluorophor increases linearly

  16. Goals • Develop a fluorescamine assay

  17. Goals • Develop a fluorescamine assay • Optimize the fluorescamine assay

  18. Goals • Develop a fluorescamine assay • Optimize the fluorescamine assay • Use the assay to assess the spin-labeling success in phospholamban

  19. Fluorescamine Assay • Measure the fluorescent intensity of fluorescamine bound to lysine, PLB, and labeled PLB in increasing concentrations to ultimately determine spin-labeling success rate

  20. Fluorescamine Assay with Lysine

  21. Fluorescamine Assay of PLB and Lysine

  22. Fluorescamine Assay with Arginine

  23. Spin Label Controls: DMF

  24. Spin Label Controls: SUCSL in DMF

  25. Fluorescamine Assay with Spin Labeled PLB

  26. Fluorescamine Assay with Spin Labeled PLB Subtracting Spin Label Control Shows 33% Labeled PLB

  27. Fluorescamine Time Control

  28. Conclusions • A lysine standard curve cannot be used to quantify PLB because of structural differences

  29. Conclusions • A lysine standard curve cannot be used to quantify PLB because of structural differences • The spin label binds to fluorescamine and forms a fluorescent compound

  30. Conclusions • A lysine standard curve cannot be used to quantify PLB because of structural differences • The spin label binds to fluorescamine and forms a fluorescent compound • Fluorescamine solution increases in fluorescence over time

  31. Future Work • Control the increasing fluorescence using a 96-well microplate reader http://openwetware.org/images/c/c7/Macintosh_HD-Users-nkuldell-Desktop-RTPCRplate.png

  32. Future Work • Apply fluorescamine assay to determine efficiency of spin-labeling PLB

  33. Future Work • Determine structure of pentameric phospholamban and further investigate how PLB interacts with the calcium pump

  34. Acknowledgements • Dr. David D. Thomas • Kurt Torgersen • The University of Minnesota • Ms. Lois Fruen • Team Research

  35. Developing a Fluorescamine Assay to Probe Cardiac Protein Structure Virginia Dines and Siddharth Damania 2006-2007

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