Comparative Proteomics Kit I: Protein Profiler Module

2. Comparative Proteomics Kit I: Protein Profiler Module

3. Protein Profiler KitInstructors Stan Hitomi Coordinator – Math & Science 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 Sherri Andrews, Ph.D. Curriculum and Training Specialist Bio-Rad Laboratories Essy Levy, M.Sc. Curriculum and Training Specialist Bio-Rad Laboratories

4. Is There Something Fishy About Teaching Evolution?Explore BiochemicalEvidence 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 ProfilerKit 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-onevolution wet lab • Sufficient materials for 8 student workstations • Can be completed in three 45 minute lab sessions

8. WorkshopTimeline • 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. LaboratoryQuick Guide

15. What’s in theSample 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? • Heating the samples denatures protein complexes, allowing the separation of individual proteins by size SDS, heat s-s Proteins with SDS – +

17. Making Proteins

18. 1o 2o 3o 4o 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 10-24 gram • Average amino acid = 110 daltons

21. 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? • Negatively charged proteins move to positive electrode • Smaller proteins move faster • Proteins separate by size SDS, heat s-s Proteins with SDS – +

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

25. 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

26. Polyacrylamide Gel Analysis Prestained Standards Actin & Myosin Sturgeon Salmon Catfish Shark Trout 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)

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

28. Determine Size of Fish Proteins Prestained Standards Actin & Myosin D A C E B 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

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

30. Molecular Mass Analysis With Semi-log Graph Paper

31. Using Gel Data to Construct a Phylogenetic Tree or Cladogram E A B C D

32. Each Fish Has a Distinct Set of Proteins

33. Some of Those Proteins Are Shared Between Fish

34. Shark Sturgeon Catfish Trout Character Matrix Is Generated and Cladogram Constructed Salmon

35. Phylogenetic Tree Evolutionary tree showing the relationships of eukaryotes. (Figure adapted from the tree of life web page from the University of Arizona (www.tolweb.org).)

36. Pairs of Fish May Have More in Common Than to the Others Shark Sturgeon Catfish Carp Salmon Trout

37. Extensions • Independent study • Western blot analysis

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