Protein Separation

1. Protein Separation BME 273 Cathy Castellon Advisor: Dr. Haselton Graduate Advisor: Greg Stone

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

3. 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)

4. 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

5. 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

6. 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

7. 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

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

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

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