1. Protein Characterization/Purification
Biochemistry Free For All

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

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

4. 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

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

6. Cation Exchange Chromatography

7. Size Exclusion / Gel Filtration Chromatography

8. Size Exclusion / Gel Filtration Chromatography

9. Affinity Chromatography

10. Reverse Phase HPLC Chromatography

11. 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.

12. 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

13. 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

14. 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

15. 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

16. Isoelectric Focusing

17. 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

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

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

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

21. 2-D Gel Electrophoresis

22. 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

23. 2-D Gel Electrophoresis

24. 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

25. 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

26. 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

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

28. 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

29. Microarray Analysis

30. Useful for identifying proteins in a gel
Western Blotting
Useful for identifying proteins in a gel

31. Proteins Must be Transferred from Gel to a Membrane
Western Blotting
Proteins Must be Transferred from Gel to a Membrane

32. Detection uses Labeled Antibody Specific to Protein of Interest
Western Blotting
Detection uses Labeled Antibody Specific to Protein of Interest