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Protein Separation Cathy Castellon BME 273 Advisor: Dr. Haselton

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Presentation on theme: "Protein Separation Cathy Castellon BME 273 Advisor: Dr. Haselton"— Presentation transcript:

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

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

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

4 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

5 Development1

6 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

7 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)

8 SDS-PAGE

9 Why a New Design? 1 Time consuming Labor intensive Resolution problems
Running the two gels can take up to 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

10 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

11 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

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

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

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

15 Reservoir 2 cm Hydrophobic Hydrophilic Flow Inlet Reservoir

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

17 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

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

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