2. Comparative Proteomics Kit I: Protein Profiler Module

3. Protein Profiler Kit Instructors
Stan Hitomi
Coordinator – Math & Science
Principal – Alamo School
San Ramon Valley Unified School District
Danville, CA
Kirk Brown
Lead Instructor, Edward Teller Education Center
Science Chair, Tracy High School
and Delta College, Tracy, CA
Bio-Rad Curriculum and Training Specialists:
Sherri Andrews, Ph.D.
Leigh Brown, M.A.
Protein Profiler Kit Instructors

4. Is There Something Fishy About Teaching Evolution
Is There Something Fishy About Teaching Evolution? Explore Biochemical Evidence for Evolution

5. Why Teach Protein Electrophoresis?
Powerful teaching tool
Real-world connections
Laboratory extensions
Tangible results
Link to careers and industry
Standards-based

7. Comparative Proteomics I: Protein Profiler Kit Advantages
Analyze protein profiles from a variety of fish
Study protein structure/function
Use polyacrylamide electrophoresis to separate proteins by size
Construct cladograms using data from students’ gel analysis
Compare biochemical and phylogenetic relationships. Hands-on evolution wet lab
Sufficient materials for 8 student workstations
Can be completed in three 45 minute lab sessions

8. Workshop Timeline Introduction Sample Preparation
Load and electrophorese protein samples
Compare protein profiles
Construct cladograms
Stain polyacrylamide gels
Laboratory Extensions

9. Traditional Systematics and Taxonomy
Classification
Kingdom
Phylum
Class
Order
Family
Genus
Species
Traditional classification based upon traits:
Morphological
Behavioral

10. Can biomolecular evidence be used to determine evolutionary relationships?

11. Biochemical Similarities
Traits are the result of:
Structure
Function
Proteins determine structure and function
DNA codes for proteins that confer traits

12. Biochemical Differences
Changes in DNA lead to proteins with:
Different functions
Novel traits
Positive, negative, or no effects
Genetic diversity provides pool for natural selection = evolution

13. Protein Fingerprinting Procedures
Day 2
Day 1
Day 3

14. Laboratory Quick Guide

15. What’s in the Sample Buffer?
• Tris buffer to provide appropriate pH
• SDS (sodium dodecyl sulfate) detergent to dissolve proteins and give them a negative charge
• Glycerol to make samples sink into wells
• Bromophenol Blue dye to visualize samples

16. Why Heat the Samples?
SDS, heat
s-s
• Heating the samples denatures protein complexes, allowing the separation of individual proteins by size
Proteins with SDS – +

18. Levels of Protein Organization

19. Protein Size Comparison
Break protein complexes into individual proteins
Denature proteins using detergent and heat
Separate proteins based on size

20. Protein Size Size measured in kilodaltons (kD)
Dalton = approximately the mass of one hydrogen atom or 1.66 x gram
Average amino acid = 110 daltons

21. Muscle Contains Proteins of Many SizeskD
Function
Titin
3000
Center myosin in sarcomere
Dystrophin
400
Anchoring to plasma membrane
Filamin
270
Cross-link filaments
Myosin
heavy chain
210
Slide filaments
Spectrin
265
Attach filaments to plasma membrane
Nebulin
107
Regulate actin assembly
-actinin
100
Bundle filaments
Gelosin 90
Fragment filaments
Fimbrin 68
Actin 42
Form filaments
Tropomysin 35
Strengthen filaments
light chain
15-25
Troponin (T.I.C.)
30, 19, 17
Mediate contraction
Thymosin 5
Sequester actin monomers
Muscle Contains Proteins of Many Sizes

22. Actin and Myosin Actin 5% of total protein
20% of vertebrate muscle mass
375 amino acids = 42 kD
Forms filaments
Myosin
Tetramer
two heavy subunits (220 kD)
two light subunits (15-25 kD)
Breaks down ATP for muscle contraction

23. How Does an SDS-PAGE Gel Work?
SDS, heat
s-s
Negatively charged proteins move to positive electrode
Smaller proteins move faster
Proteins separate by size
Proteins with SDS – +

24. Chemistry in action…. Or… Ask your friendly chemist… about electro-phoresis and electrolysis.
The very basic concepts of gel electrophoresis and electrolysis

25. Chemistry of electrophoresis and electrolysis Electric fields and electric currents
Charged particles migrate toward the opposite charge in an electric field. This is the basis of electrophoresis.

26. SDS-PAGE, DNA electrophoresis and the need for a gel matrix (sieving medium) Without a viscous medium, all molecules move at ~ the same rate in electric field Gel structure retards larger solute particles DNA molecules, SDS- proteins have ~ equal charge/mass Electrophoresis occurs between the electrodes (field-driven).
In a sieving medium, size matters. Even with a constant charge/mass ratio, larger particles encounter a larger force resisting their motion, and therefore their progress is retarded relative to smaller particles.
In a non-viscous medium (free solution approximates this, and a vacuum, as in a mass spectrometer, is the ideal,) all particles with the same ratio of mass to charge move equally.

27. Electrolysis always occurs during electro-phoresis
Electrolysis always occurs during electro-phoresis. Cathode produces H2 at twice the rate that anode produces O2 Current is carried by solute ions. Electrons aren’t soluble in H2O. Example: TAE buffer; tris supplies cations (+), acetate supplies anions (-). Electrolysis occurs at the electrodes.
At the cathode, the hydrogen ion, which is a hydrated proton, takes an electron to become a neutral hydrogen atom. Two hydrogen atoms bond covalently to become a hydrogen molecule (gas) which bubbles to the surface.
The anode reaction strips 4 electrons from 2 water molecules, freeing soluble protons, and giving 2 oxygen atoms, which bond covalently to give an oxygen molecule, which bubbles to the surface.

28. SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
CH2
CH3
SDS
SDS detergent (sodium dodecyl sulfate)
Solubilizes and denatures proteins
Adds negative charge to proteins
Heat denatures proteins

29. Chemistry in action…. Or… Ask your friendly chemist… about detergents.
Basic overview of detergent broad classes.

30. DETERGENTS:… are amphiphiles, containing a lipophilic portion and a hydrophilic portion. lower the interfacial energy between unlike phases. emulsify or solubilize aggregated particles.
I like fat!
Detergents are broadly classified according to the intensity of their interactions with tissues, cells, membranes and macromolecules.
I like water!

31. More about detergent terms Lipophilic portion is also referred to as “hydrophobic” tail Hydrophilic portion is also referred to as “polar” head Types: nonionic, anionic, cationic and zwitterionic
The common feature among detergents is the combination of a lipophilic and a hydrophilic portion. The types vary in the nature of the two portions. Common detergents tend to aggregate, or micellize once they reach a certain concentration in a given buffer system. The aggregates behave in ways that single molecules don’t, having an ordered supramolecular structure.

32. Detergents: Ionic vs non-ionic Denaturing vs non-denaturing Swords (denaturing): “pointy” hydrophobic ends, ionic polar ends Gloves (non-denaturing): bulky, non-penetrating hydrophobic ends, non-ionic or zwitterionic polar ends.
SDS
Triton X-100
Denaturing detergents disrupt cellular aggregates, membranes, protein complexes and the structures of proteins themselves, because their shape allows them to penetrate structures that water or buffers alone cannot reach. Detergents lower interfacial tension to varying degrees.
Non-denaturing detergents are structurally bulkier in their hydrophobic portions, and cannot fit into hydrophobic folds in proteins as well as denaturing detergents can. They can still emulsify and separate many protein complexes and cellular aggregates, but leave many proteins functionally intact. They interfere with low-specificity interactions, such as aggregation of proteins, but do not generally interfere with high-specificity interactions, such as antibody-antigen or enzyme-substrate binding, unless they happen to mimic the structure of the antigen or substrate.

33. Why Use Polyacrylamide Gels to Separate Proteins?
Polyacrylamide gel has a tight matrix
Ideal for protein separation
Smaller pore size than agarose
Proteins much smaller than DNA
Average amino acid = 110 daltons
Average nucleotide pair = 649 daltons
1 kilobase of DNA = 650 kD
1 kilobase of DNA encodes 333 amino acids = 36 kD

34. Polyacrylamide Gel Analysis
Prestained
Standards
Actin & Myosin
Sturgeon
Shark
Salmon
Trout
Catfish
250
150
100
Myosin Heavy Chain (210 kD) 75 50 37
Actin (42 kD)
Tropomyosin (35 kD) 25 20
Myosin Light Chain 1 (21 kD) 15
Myosin Light Chain 2 (19 kD) 10
Myosin Light Chain 3 (16 kD)

35. Can Proteins be Separated on Agarose Gels?20 25 37 50 75
100
150
250
Prestained
Standards
Shark
Salmon
Trout
Catfish
Sturgeon
Actin & Myosin
Myosin Heavy Chain
Actin
Tropomyosin
Myosin Light Chains
Prestained
Standards
Shark
Salmon
Trout
Catfish
Sturgeon
Actin & Myosin
Myosin Heavy Chain
Actin
Tropomyosin 10 15 20 25 37 50 75
100
150
250
Myosin Light Chains
Polyacrylamide
Agarose

36. Determine Size of Fish Proteins
Prestained
Standards
Actin & Myosin A B C D E
Measure distance from base of wells to the base of the bands
250
150
100 75 50 37 25
Measure prestained standard bands between ~30 and 10 kD 20
Measure fish protein bands between ~30 and 10 kD 15 10

37. Molecular Mass Estimation
37 (12 mm)
25 (17 mm)
20 (22 mm)
15 (27.5 mm)
10 (36 mm)

38. Molecular Mass Analysis With Semi-log Graph Paper

39. Using Gel Data to Construct a Phylogenetic Tree or CladogramB C D E

40. Each Fish Has a Distinct Set of Proteins
Shark
Salmon
Trout
Catfish
Sturgeon
Total # proteins 8 10 13 12
Distance proteins migrated (mm)
25, 26.5, 29, 36, 36.5, 39, 44, 52
26, 27.5, 29, 32, 34.5, 36.5, 37.5, 40.5, 42, 45
26, 27.5, 29, 29.5, 32, 34.5, 36.5, 37.5, 40.5, 42, 45, 46.5, 51.5
26, 27.5, 29, 32, 36.5, 38, 38.5, 41, 46, 47.5
26, 27.5, 30, 30.5, 33, 35.5, 37, 39, 39.5, 42, 44, 47

41. Some of Those Proteins Are Shared Between Fish
Distance (mm)
Size (kD)
Shark
Salmon
Trout
Catfish
Sturgeon 25
32.5 X 26
31.5
26.5
31.0
27.5
30.0
28.5
29.1 29
28.6 30
27.6
30.5
27.1 32
25.6 33
24.7
34.5
23.2
35.5
22.2 36
21.7
36.5
21.2 37
20.7
37.5
20.2 38
19.7
38.5
19.3
Some of Those Proteins Are Shared Between Fish

42. Character Matrix Is Generated and Cladogram Constructed
Shark
Salmon
Trout
Catfish
Sturgeon 8 2 10 5 3 13 4 12
Character Matrix Is Generated and Cladogram Constructed
Shark
Sturgeon
Catfish
Trout
Salmon

43. Phylogenetic Tree
Evolutionary tree showing the relationships of eukaryotes.
(Figure adapted from the tree of life web page from
the University of Arizona (

44. Pairs of Fish May Have More in Common Than to the Others
Shark
Salmon
Trout
Catfish
Sturgeon
Carp 8 2 10 5 3 13 4 12 11
Pairs of Fish May Have More in Common Than to the Others
Shark
Sturgeon
Catfish
Carp
Trout
Salmon

45. Extensions
Independent study
Western blot analysis

46. Ready Gel® Precast Gel Assembly
Step 1
Step 2
Step 3
Step 4

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