Comparative Proteomics Kit I: Protein Profiler Module

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. sherri_andrews@bio-rad.com Leigh Brown, M.A. leigh_brown@bio-rad.com Protein Profiler Kit Instructors

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

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

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

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

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

Can biomolecular evidence be used to determine evolutionary relationships?

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

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

Protein Fingerprinting Procedures Day 2 Day 1 Day 3

Laboratory Quick Guide

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

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 – +

Levels of Protein Organization

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

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

Muscle Contains Proteins of Many Sizes 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 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

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

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 – +

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

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.

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.

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.

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

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

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!

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.

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.

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

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)

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

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

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

Molecular Mass Analysis With Semi-log Graph Paper

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

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

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

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

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).)

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

Extensions Independent study Western blot analysis

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

Webinars Enzyme Kinetics — A Biofuels Case Study Real-Time PCR — What You Need To Know and Why You Should Teach It! Proteins — Where DNA Takes on Form and Function From plants to sequence: a six week college biology lab course From singleplex to multiplex: making the most out of your realtime experiments explorer.bio-rad.comSupportWebinars