Protein Separation Cathy Castellon BME 273 Advisor: Dr. Haselton

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Presentation transcript:

Protein Separation Cathy Castellon BME 273 Advisor: Dr. Haselton Graduate Advisor: Greg Stone 11/29/2018

Overview of Proteins1 Master molecules of living things Composition Development (central dogma)

Master Molecules of Living Things1 Enzymes Structural proteins Contractile proteins Membrane proteins Transport proteins Storage proteins Protective proteins Hormones

Composition1 Proteins are polymers of amino acids Each amino acid has an alpha carbon with four groups attached Carboxylic acid group (COOH) Amine group Hydrogen atom An “R”group that make it distinct from other amino acids Amino acids combine to form peptides through peptide bonds Proteins consist of one or more peptides

Development1

Improving Protein Separation Current Technology 2D protein electrophoresis(SDS-PAGE) The Need 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 Economics Faster more efficient technology

How does 2DE work? 1 Separates proteins by isoelectric point (pI) And second by size (molecular weight) using sodium didecyl sulfate polyacrylamide gel electorphoresis (SDS-PAGE)

SDS-PAGE

Why a New Design? 1 Time consuming Labor intensive Resolution problems Running the two gels can take up to 10-15 hrs Labor intensive Gels are often hand cast Samples must be loaded by hand Tube gel must be properly combined with the slab gel Resolution problems Usually Less then 1000 proteins can be resolved

Our Thoughts Design Microfluid Technique Separation based on hydrophobicity 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

Game Plan In the Works Accomplished Flurometer Setup Prototype Slides gradient contact angle measurements Simple Channel Design Lithograph technique PDMS Detailed Channel Design Mix protein Protein Labeling In the Works Flurometer Setup Prototype

Slides Gradient Contact Angle Measurements PDMS Adherence hydro-phobic/phyllic Contact Angle Measurements PDMS Adherence

Simple Channel Design Lithography Technique Single 2X2 cm Lane PDMS Works with clean slide Doesn’t work (yet) with hydrophobic slide Claming the prototype

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

Reservoir 2 cm Hydrophobic Hydrophilic Flow Inlet Reservoir

Protein Labeling 2 different proteins 2 different labels Cytocrsome C and Lysosyme 2 different labels AlexaFluro 430 (540nm) and 350 (442nm)

Furture Work: Setup Flow Chamber/ Fluorescence Spectrophotometer Flow Chamber-basic idea monitor pressure of flow monitor flow rate Spectrophotometer test each reservoir and measure labeled protein signal

References Protein Notes- Greg Stone (1) Lithograph Technique- Dave Schaffer Mass Spectrometry (SDS-PAGE)- Dr. David Hachey