New Technology for Protein Separation

BME 273

Cathy CastellonAdvisor: Dr. Rick Haselton

Graduate Advisor: Greg Stone

Protein Background Master Molecules of

Living Things Composition Central Dogma

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

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

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

Lithography Technique Coat Substrate with

Photoresist Apply Mask/ Expose

Photoresist to Light Develop Photoresist Cast and Cure

PDMS Remove PDMS from

Substrate

Detailed Channel Design

2X2 cm lanes Hydro-phobic/phyllic

on same slide (R,L) Posts used to aid

mixing and accentuate the separation

Slides Gradient

– hydro-phobic/phyllic

Contact Angle Measurements

PDMS Adherence

3-glicidoxypropyltrimethoxysilane

octyltrichlorosilane

Strategy for Prototype 2 different proteins

– CytochromeC and Lysozyme

2 different labels – AlexaFluro 430 (540nm) and 350 (442nm)

Cytochrome CLysozyme

Unforeseen Problems

Si-Lanes lost reactivity– Gradient could not be improved

Micro-fluid channel– leak

HPLC column – Incorrect size

Future Work

Flow Chamber-basic idea– monitor pressure of flow – monitor flow rate

Spectrophotometer– test each reservoir and measure labeled

protein signal

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