Protein Characterization/Purification Biochemistry Free For All

Protein Purification Opening Cells Centrifugation Fractionation Applications of Biochemistry Knowledge Opening Cells Centrifugation Fractionation

Dialysis Separates salts from proteins Applications of Biochemistry Knowledge Separates salts from proteins

Chromatography (Column) Applications of Biochemistry Knowledge Separation based on charge - Ion Exchange Separation based on size - Size Exclusion / Gel Filtration Separation based on affinity - Affinity Chromatography Separation based on polarity - Reverse Phase Chromatography

Ion Exchange Chromatography Cation exchange chromatography (+ sticks) Anion exchange chromatography (- sticks)

Cation Exchange Chromatography

Size Exclusion / Gel Filtration Chromatography

Size Exclusion / Gel Filtration Chromatography

Affinity Chromatography

Reverse Phase HPLC Chromatography

Reverse Phase HPLC Chromatography Columns have non-polar packing material Non-polar materials interact more with column than polar materials The most polar materials will elute first. The most non-polar materials will elute last.

Agarose Gel Electrophoresis Mesh-like support Evenly charged rod-like molecules (negative) Samples loaded in wells Electrical current pushes molecules through support All molecules have same mass to charge ratio Largest molecules move slowest

Agarose Gel Electrophoresis Mesh-like support Evenly charged rod-like molecules (negative) Samples loaded in wells Electrical current pushes molecules through support Largest molecules move slowest Loading Wells Largest Smallest

Agarose Gel Electrophoresis Mesh-like support Evenly charged rod-like molecules (negative) Samples loaded in wells Electrical current pushes molecules through support Largest molecules move slowest

Polyacrylamide Gel Electrophoresis Mesh-like support - tinier pores Negatively charged rod-like molecules (SDS-protein) Samples loaded in wells Electrical current pushes molecules through support All molecules have same mass to charge ratio Largest molecules move slowest Largest Smallest

Isoelectric Focusing

Proteomics - 2D Gel Electrophoresis In Proteomics, Researchers Aim to Quantitate All of the Proteins Made in Cell/Tissue 2-D Gel Electrophoresis is One Way to Do This Analysis

Proteomics - 2D Gel Electrophoresis Add Protein Mixture to Polyelectrolyte Column

Proteomics - 2D Gel Electrophoresis Apply Electrical Current Proteins Separate According to pI Values High pI Low pI

Proteomics - 2D Gel Electrophoresis Separated By Charge/pI Rotate Apply to Gel Add SDS Separate By Size on Polyacrylamide Gel

2-D Gel Electrophoresis

Proteomics - 2D Gel Electrophoresis Separated By Charge/pI Each Spot Corresponds to a Unique Protein Separated By Size The Intensity of Each Spot is a Measure of the Amount of Protein Present

2-D Gel Electrophoresis

Biotechnology Take Two Sets of Cells - Healthy vs Cancerous Proteomics Take Two Sets of Cells - Healthy vs Cancerous Label Proteins Orange Label Proteins Blue Orange - Proteins in a Healthy Cell, But Not a Cancer Cell Blue - Proteins in a Cancer Cell, But Not a Healthy Cell Black - Proteins Equally Abundant in Both Cells

Microarray Analysis Gene #3 Gene #2 Microarray Analysis Allows a Researcher to Measure the Quantity of every mRNA of Interest Made in a Cell/Tissue Gene #1 Chemically Synthesize DNA Corresponding to an mRNA Bond Thousands of Copies of That DNA to a Spot on a Slide Repeat for Every mRNA of an Organism One Spot Per mRNA

Microarray Analysis Take Two Sets of Cells - Healthy vs Cancerous Isolate All mRNAs from Each Copy Each mRNA Using Reverse Transcriptase to Make cDNA Copies of Each Add Fluorescent Green Tag to Normal Cell cDNAs Add Fluorescent Red Tag to Cancer Cell mRNAs

Microarray Analysis Mix cDNA Samples Pour Mixture Onto Slide Allow Hybridization to Occur Wash Unhybridized Samples Away

Microarray Analysis Intensity of Color Measures Amount of mRNA Shade of Color Measures Relative Expression Between Cell Types Bright Green - Abundant In Healthy Cells, Not in Cancer Cells Bright Red - Abundant In Cancer Cells, Not in Healthy Cells Black - Absent in Both Cells Types Bright Yellow - Abundant In Both Cells Types

Microarray Analysis

Useful for identifying proteins in a gel Western Blotting Useful for identifying proteins in a gel https://en.wikipedia.org/wiki/Western_blot#/media/File:SDS-PAGE_sample.png

Proteins Must be Transferred from Gel to a Membrane Western Blotting Proteins Must be Transferred from Gel to a Membrane https://en.wikipedia.org/wiki/Western_blot#/media/File:Western_blot_transfer.png

Detection uses Labeled Antibody Specific to Protein of Interest Western Blotting Detection uses Labeled Antibody Specific to Protein of Interest https://en.wikipedia.org/wiki/Western_blot#/media/File:Western_Blot_binding.png