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Isolation and Characterization of Cellular Complexes Containing the Histone Deacetylase SIRT1

Isolation and Characterization of Cellular Complexes Containing the Histone Deacetylase SIRT1. Vincent Lew Howard Hughes Medical Institute Fellowship Mentor: Dr. Mark Leid Molecular Pharmacology Lab College of Pharmacy - Oregon State University. DNA.

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Isolation and Characterization of Cellular Complexes Containing the Histone Deacetylase SIRT1

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  1. Isolation and Characterization of Cellular Complexes Containing the Histone Deacetylase SIRT1 Vincent Lew Howard Hughes Medical Institute Fellowship Mentor: Dr. Mark Leid Molecular Pharmacology Lab College of Pharmacy - Oregon State University

  2. DNA • Humans have 14,000 to 73,000 micrometers of DNA per chromosome • All 46 chromosomes together in a human cell have 2 meters of DNA which is contained in a nucleus that is 5 micrometers in diameter How does it fit?

  3. - - + + How does it fit?

  4. History • Human Jurkat Cells (Immune System) • Silent Information Regulator (SIRT) • Chicken (Ovalbumin Upstream Promoter) Transcription Factor Interacting Proteins 1 & 2 (CTIP1 & CTIP2) • Histone DeAcetylase Complex (HDAC) Represses Gene Transcription

  5. Project Aim • To isolate Silent Information Regulator (SIRT) protein & complexes via purification • To investigate all of the functions of SIRT in the human body • Determine by finding what proteins are found with it

  6. ? ? SIRT ?

  7. RNA Polymerase RNA Polymerase Acetylation Complex Acetylation Complex Activation Acetylation Complex

  8. RNA Polymerase CTIP / SIRT Complex CTIP / SIRT Complex Repression

  9. Cl- Cl- Na+ Na+ Step One: Phosphocellulose IN • Phosphocellulose Column • Tightly packed resin • Negatively charged • Wash w/ increasing NaCl molarity (stronger washes) COLLECT

  10. Step Two: Where is the Protein? • Electrophoresis • Negatively Charged & Migrates to Cathode • Smaller strands migrate faster - 10% Acrylamide Gel +

  11. Step Two Continued • Electrotransfer • Use of electricity to transfer proteins from acrylamide gel to nitrocellulose membrane Nitrocellulose Membrane 10% Acrylamide Gel

  12. chemiluminescence Step Three: Western Blot • Immunoreactivity • Use of antibodies that bind to specific proteins specific protein nitrocellulose membrane

  13. Western Blot Results Flow Through 300 mM 600 mM Input • What does this mean? • There is a significant amount of SIRT protein in the Input & Flow Through • Input: originally put into the column (before purification) • Flow Through: protein that did not stick to the column

  14. Cl- Cl- Na+ Na+ Step Four: DEAE IN • Diethylaminoethyl Column • Tightly packed resin • Positively charged • Wash w/ increasing NaCl molarity (stronger washes) COLLECT

  15. Western Blot Results Flow Through 400 mM 800 mM Input 600 mM 1 M 200 mM • What does this mean? • There is SIRT protein in the Input, .2mM & .4mM lanes • Input: originally put into the column (before purification) • .2mM: what eluted after .2 milli-Molar NaCl solution • .4mM: what eluted after .4 milli-Molar NaCl solution

  16. Step Six: Size Exclusion • Size Exclusion column • Separates based on SIZE of molecules • Use of known markers to determine size of unknown molecules • Thyroglobulin: 669 kD • Catalayse: 232 kD • BSA (albumin): 67 kD • 1 kiloDalton = 1.67 x 10-24 kg

  17. Size Exclusion IN OUT

  18. Western Blot Results Thy (669) Cat (232) BSA (67) 200 mM Thy (669) Cat (232) 400 mM

  19. Step Seven: Immunoprecipitate • Final step of purification in this project • Use of specific antibody (Abx) to bind to individual protein complex • Extract only that which is bound to antibody

  20. Sepharose Beads Sir2 Abx Protein X Protein Y SIRT SIRT complexed to unknown protein Immunoprecipitation

  21. Mass Spectrometry • Determine protein structure based on molecular mass

  22. Results • Still to be determined

  23. Acknowledgements • Howard Hughes Medical Institute • Dr. Mark Leid – Molecular Pharmacology, Oregon State University • Valerie Peterson – Molecular Pharmacology, Oregon State University

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