Comparative Proteomics Kit I: Protein Profiler Module
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2 Comparative Proteomics Kit I: Protein Profiler Module
3 Protein Profiler Kit Instructors 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 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
6
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? s-s SDS, heat Heating the samples denatures protein complexes, allowing the separation of individual proteins by size Proteins with SDS +
17 Making Proteins DNA TAC GGA TCG AGA TGA mrna AUG CCU AGC UCU ACU trna UAC GGA UCG AGA UGA Amino Acid Tyr Gly Ser Arg STOP
18 Levels of Protein Organization 1 o 2 o 3 o 4 o
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 Sizes Protein kd 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 Bundle filaments Actin 42 Form filaments Tropomysin 35 Strengthen filaments Myosin light chain Slide filaments Troponin (T.I.C.) 30, 19, 17 Mediate contraction Thymosin 5 Sequester actin monomers
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? s-s SDS, heat Negatively charged proteins move to positive electrode Smaller proteins move faster Proteins separate by size + Proteins with SDS
24 SDS- Polyacrylamide Gel Electrophoresis (SDS-PAGE) CH 3 CH 2 CH 2 CH 2 CH 2 CH 2 SDS detergent (sodium dodecyl sulfate) Solubilizes and denatures proteins Adds negative charge to proteins Heat denatures proteins CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 O O S O - O SDS
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 Myosin Heavy Chain (210 kd) Actin (42 kd) Tropomyosin (35 kd) Myosin Light Chain 1 (21 kd) Myosin Light Chain 2 (19 kd) Myosin Light Chain 3 (16 kd)
27 Can Proteins be Separated on Agarose Gels? Myosin Heavy Chain Actin Tropomyosin Myosin Light Chains Myosin Heavy Chain Actin Tropomyosin Myosin Light Chains 10 Polyacrylamide Agarose
28 Determine Size of Fish Proteins Measure prestained standard bands between ~30 and 10 kd Measure distance from base of wells to the base of the bands Measure fish protein bands between ~30 and 10 kd
29 Molecular Mass Estimation (12 mm) 25 (17 mm) 20 (22 mm) 15 (27.5 mm) 10 (36 mm) Distance migrated (mm)
30 Size (kd) Molecular Mass Analysis With Semi-log Graph Paper Distance migrated (mm)
31 Using Gel Data to Construct a Phylogenetic Tree or Cladogram A B C D E
32 Each Fish Has a Distinct Set of Proteins Total # proteins Distance proteins migrated (mm) Shark Salmon Trout Catfish Sturgeon 25, 26.5, 29, 36, 36.5, 39, 44, , 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, , 27.5, 29, 32, 36.5, 38, 38.5, 41, 46, , 27.5, 30, 30.5, 33, 35.5, 37, 39, 39.5, 42, 44, 47
33 Some of Those Proteins Are Shared Between Fish Distance (mm) Size (kd) Shark X Salmon X X X X X X X X X Trout X X X X X X X X X X X X X X X X X X X X X X X X Catfish Sturgeon
34 Character Matrix Is Generated and Cladogram Constructed Shark Salmon Trout Catfish Sturgeon Shark Salmon Trout Catfish Sturgeon Shark Sturgeon Catfish Trout 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 (
36 Pairs of Fish May Have More in Common Than to the Others Shark Salmon Trout Catfish Sturgeon Carp Shark Salmon Trout Catfish Sturge on Carp Shark Sturgeon Catfish Carp Trout Salmon
37 Extensions Independent study Western blot analysis
38 Ready Gel Precast Gel Assembly Step 1 Step 2 Step 3 Step 4
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