Protein Separation Cathy Castellon BME 273 Advisor: Dr. Haselton Graduate Advisor: Greg Stone 9/18/2018

Proteomics: Current Technology The need and desire to understand total protein expression Relies on the microchemical characterization of peptides separated by 2D protein electrophoresis.

How does 2DE work? Separates proteins by isoelectric point (pI) And second by size (molecular weight) using sodium didecyl sulfate polyacrylamide gel electorphoresis (SDS-PAGE)

Application of 2DE 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 Example: Exposure of rat kidney to lead alters: 76 proteins in cortex 13 proteins in medulla Separate complex protein mixtures into their individual polypeptide components

Improvements Mass Spectrometry with isotope labeling Using isotope-coded affinity tags Molecular Scanner takes 2DE gels and combines protease digestion and electroblotting to a membrane in a single step

Our Thoughts Microfluid Technique Uniformly hydrophobic slide Create flow channel (lithography) Create inlet and outlet points Load fluorescently labeled protein solution into one end Pump buffer solution through the channel Fluoremeter will detect separation

Current Work Produce hydrophobic/phillic gradient slides Use Si-lane glass slides Measure Contact Angles This will enable us to determine the hydrophobicity of a particular protein

Slide III (1-25-2002) Hydrophobic gradient 3 4 6 5 1b 2b 3b 4b 5b 6b

Current Plan Microfluid Chamber shallow and relatively wide increase surface area interaction ridges that mimic chamber below

Future Work Come up with a simple experiment to pretest our assumptions. Create gradient Create microchannel mix protein solution back and forth fluorescent scanner