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New Technology for Protein Separation

This project explores a cutting-edge microfluidic approach to protein separation, aiming to automate and optimize existing methods that are often time-consuming and labor-intensive. By adapting techniques such as soft lithography, the design includes a flow channel that separates proteins based on their hydrophobicity while significantly increasing throughput—up to 100-fold. The prototype will facilitate the detection of fluorescently labeled proteins, offering insights into protein profiles under different conditions. Future work includes monitoring flow rates and optimizing channel gradients to enhance resolution.

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New Technology for Protein Separation

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  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

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