1 / 11

New Technology for Protein Separation

New Technology for Protein Separation. BME 273 Cathy Castellon Advisor: Dr. Rick Haselton Graduate Advisor: Greg Stone. Protein Background.

vanna-rodgers
Download Presentation

New Technology for Protein Separation

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. New Technology for Protein Separation BME 273 Cathy Castellon Advisor: Dr. Rick Haselton Graduate Advisor: Greg Stone

  2. Protein Background • To compare the expression of protein profiles from an arbitrary reference state of a cell, tissue, or organism, to the profile of an non-standard condition • Master Molecules of Living Things • Composition • Central Dogma

  3. How Does Current Technology Work? • Separates proteins by: • isoelectric point (pI) • size (molecular weight) • Problems • large volume • time consuming • resolution problems • labor intensive • Economics • Need a faster more efficient technology that will separate proteins 100-fold at once

  4. Design Goals • Adapt Micro-fluid Technique • Create flow channel (soft lithography) • Separation based on hydrophobicity • Create inlet and outlet points • Load fluorescently labeled protein solution into one end • Pump buffer solution through the channel • Fluoremeter will detect separation • Automate

  5. Lithography Technique • Coat Substrate with Photoresist • Apply Mask/ Expose Photoresist to Light • Develop Photoresist • Cast and Cure PDMS • Remove PDMS from Substrate

  6. Detailed Channel Design • 2X2 cm lanes • Hydro-phobic/phyllic on same slide (R,L) • Posts used to aid mixing and accentuate the separation

  7. Slides 3-glicidoxypropyltrimethoxysilane octyltrichlorosilane • Gradient • hydro-phobic/phyllic • Contact Angle Measurements • PDMS Adherence

  8. Strategy for Prototype Lysozyme Cytochrome C • 2 different proteins • CytochromeC and Lysozyme • 2 different labels • AlexaFluro 430 (540nm) and 350 (442nm)

  9. Unforeseen Problems • Si-Lanes lost reactivity • Gradient could not be improved • Micro-fluid channel • leak • HPLC column • Incorrect size

  10. Future Work • Flow Chamber-basic idea • monitor pressure of flow • monitor flow rate • Spectrophotometer • test each reservoir and measure labeled protein signal

  11. References • DoInik, Vladislav, Shaorong Liu, and Stevan Jovanovich. Capillary electrophoreses on microchip. Electrophoresis 2000. 21, 41-54. • Stroock, Abraham D., Stephan K.W. Dertinger, etal. Chaotic Mixer for Microchannels. Science. Vol 295, 647-651. • Hopp, Thomas P. and Kenneth R. Woods. Prediction of Protein Antigenic Determinants from Amino Acid Sewquences. National Academy of Sciences of the USA. Vol. 78, Issue 6, 3824-3828. • http://www.sdk.co.jp/shodex/english/dc010603.htm • http://mstflab.vuse.vanderbilt.edu/projects/microfluidics/soft_lithography_intro.html • http://www.unitedchem.com/1024x768/Uct2.htm • http://metallo.scripps.edu/PROMISE/1BBH.html • http://www.rcsb.org/pdb/molecules/pdb9_1.html • http://www.worthington-biochem.com/manual/L/LY.html • http://crystal.uah.edu/~carter/protein/xray.htm • Acknowledgements • Dr. Rick Haselton, Advisor, Vanderbilt University • Greg Stone, Graduate Student, Vanderbilt University • David Schaffer, Graduate Student, Vanderbilt University • Dr. David Hachey, Mass Spectrometry Vanderbilt University

More Related