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

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Is There Something Fishy About Teaching
Evolution?Explore Biochemical Evidence for
Evolution
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Protein Fingerprinting Kit
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Protein Fingerprinting KitInstructors

• Stan Hitomi
• Director, Edward Teller Education Center
• UC Davis / Lawrence Livermore National
• Laboratory, Livermore, CA
• Kirk Brown
• Lead Instructor, Edward Teller Education Center
• Science Chair, Tracy High School
• and Delta College, Tracy, CA
• Sherri Andrews, Ph.D.
• North Carolina School of the Arts
• Winston-Salem, NC

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Why Teach Protein Electrophoresis?

• Powerful teaching tool
• Real-world connections
• Laboratory extensions
• Tangible results
• Link to careers and industry

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Hands-on Evolution Wet Lab

• Analyze protein profiles from a variety of fish
• Use acrylamide electrophoresis to separate
  proteins by size
  
• Compare biochemical and phylogenetic
  relationships
  

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Traditional Systematics and Taxonomy

• Classification
• Kingdom
• Phylum
• Class
• Order
• Family
• Genus
• Species
• Traditional classification based upon traits
• Morphological
• Behavioral

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Can biomolecular evidence be used to determine
evolutionary relationships?
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Biochemical Similarities

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

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Biochemical Differences

• Changes in DNA leads to proteins with
• Different functions
• Novel traits
• Positive, negative or no effects
• Genetic diversity provides pool for natural
  selection evolution
  

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Protein Fingerprinting Procedures

• Day 2

Day 3
Day 1
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Whats 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

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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


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Making Proteins
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Levels of Protein Organization
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Protein Size Comparison

• Break protein complexes into individual proteins
• Denature proteins using detergent and heat
• Separate proteins based on size

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Protein Size

• Size measured in kilodaltons (kDa)
• Dalton mass of hydrogen molecule
• 1.66 x 10-24 gram
• Average amino acid 110 daltons

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Muscle Contains Proteins of Many Sizes
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Actin and Myosin

• Actin
• 5 of total protein
• 20 of vertebrate muscle mass
• 375 amino acids 42 kDa
• Forms filaments
• Myosin
• Tetramer
• two heavy subunits (220 kDa)
• two light subunits (20 kDa)
• Breaks down ATP for muscle contraction

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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


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SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

• SDS Detergent (Sodium Dodecyl Sulfate)
• Solubilizes and denatures proteins
• Adds negative charge to proteins
• Heat denatures proteins

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Why Use Acrylamide Gels to Separate Proteins?

• Acrylamide gel give a tight matrix
• Ideal for protein separation
• Smaller pore size than agarose
• Proteins much smaller than DNA
• Average amino acid 110 Da
• Average nucleotide pair 649 Da
• 1 kilobase of DNA 650 kDa
• 1 kilobase of DNA encodes 333 amino acids 36
  kDa
  

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Gel Analysis

• Lane
• Kaleidoscope Markers
• Shark
• Salmon
• Trout
• Catfish
• Sturgeon
• Actin and Myosin Standard

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Molecular Weight Estimation
250
200
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Size in kDa
100
50
0
0
20
40
60
Distance (mm from well)
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Molecular Weight Analysis
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Phylogenetic Tree
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Extensions

• Independent Study
• Western blot analysis

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Ready Gel Assembly

• Step 1

Step 2
Step 3
Step 4