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Protein Fingerprinting and Evolution. Ms. Haut. Protein Fingerprinting. Analyze protein profiles from a variety of fish Use acrylamide electrophoresis to separate proteins by size Compare biochemical and phylogenetic relationships. Making Proteins. DNA TACGGATCGAGATGA - PowerPoint PPT Presentation
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Analyze protein profiles from a variety of fish
Use acrylamide electrophoresis to separate proteins by size
Compare biochemical and phylogenetic relationships
Protein Fingerprinting
Making Proteins
DNA TAC GGATCG AGATGA
mRNA AUGCCUAGCUCUACU
tRNA UACGGAUCGAGAUGA
Amino Acid Tyr Gly Ser Arg STOP
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 (kDa)
Dalton = mass of hydrogen molecule
= 1.66 x 10-24 gram
Average amino acid = 110 daltons
Muscle Contains Proteins of Many Sizes
Protein kDa 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
27 Slide filaments
Troponin (T.I.C) 30, 19, 17 Mediate contraction
Thymosin 5 Sequester actin monomers
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
Campbell, 2007.
Protein Fingerprinting First carried out in 1956 to show that
sickle-cell anemia was caused by a change in a single amino acid of the hemoglobin protein
The method we will use SDS-PAGE was developed in 1970 by U.K. Laemmli
Protein Electrophoresis
Proteins are charged molecules whose sequences are determined by DNA
Polyacrylamide gels are used to separate small molecules such as proteins Agarose gels separate large molecules
The percentage of acrylamide gel to be used depends on the size range of the proteins of interest. (the higher the % = the denser the matrix and better the separation of small molecules)
http://departments.oxy.edu/biology/Franck/Bio222/Lectures/Feb1lecture.htm
Protein Electrophoresis
Proteins are separated by size—smaller proteins travel farther in the matrix
Useful Information Gathered from Protein Sequence Relationship to other proteins (protein families)
Example: Viral protein that produces cancer is nearly identical to normal cellular growth factor.
Evolution of organisms (phylogenetic trees).
Information for creating antibodies: specific regions can be identified.
Information for making DNA probes.
Traditional Systematics and Taxonomy Classification
Kingdom Phylum Class Order Family Genus Species
Traditional classification based upon traits: Morphological Behavioral
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 leads to proteins with: Different functions Novel traits Positive, negative or no
effects
Genetic diversity provides pool for natural selection = evolution
Evolution and Classification of Fishes Evolutionary
trees Show
evolutionary lineages of different species over time
http://bio.winona.edu/berg/ILLUST/evoltree.gif
Protein gel electrophoresis
Protein gel from protein fingerprinting experiment. Each blue band represents a distinct protein. The pattern of bands gives information about the composition of a sample
http://www.bridgewater.edu/~sbaron/Bio%20325%20Pics.htm
Species Sequence
Beef, Horse, Pig .......val.gln.lys.cys.ala.gln.cys.his.thr.val.glu.lys....
Salmon .......val.gln.lys.cys.ala.gln.cys.his.thr.val.glu.....
Chicken .......val.gln.lys.cys.ser.gln.cys.his.thr.val.glu.....
Silkworm .......val.gln.arg.cys.ala.gln.cys.his.thr.val.glu.....
Yeast phe.lys.thr.arg.cys.glu.leu.cys.his.thr.val.glu.....
Rhodospirillum rubrum
lys.cys.leu.ala.cys.his.thr.phe.asp.glu.gly.ala.asp.lys....
Common Sequence --------------.....cys.X.Y.cys.his.thr.......
Variations in amino acid sequence in one part of the cytochrome c molecule
Variations in amino acid sequence in one part of the cytochrome c molecule
http://www.rtis.com/nat/user/elsberry/evobio/evc/argresp/sequence.html
Fish Classification Chondrichthyes (cartilaginous fishes)
Cartilaginous skeleton, thick skin w/o scales, no swim bladders or lungs
Sharks, skates, rays
Osteichthyes (bony fishes) Bony skeleton, true scales, paired fins w/ moveable
rays
Agnatha (jawless fishes) Eel-like, jawless fishes with parasitic and scavenging
lifestyles No scales or paired fins
http://www.fao.org/documents/show_cdr.asp?url_file=/DOCREP/V7180E/v7180e04.htm
Phylogenetic tree for fishes
Which fishes should you select?
Choose fish specimens that will provide striking and distinct results
Select some closely related fishes (salmon/trout) and some more distantly related fishes (get exotic—shark, tuna)
Other aquatic organisms—mollusks—scallops, octopus; arthropods—clams, oysters, crab, shrimp
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
http://departments.oxy.edu/biology/Franck/Bio222/Lectures/Feb1lecture.htm
Why Heat the Samples?
• Heating the samples denatures protein complexes, allowing the separation of individual proteins by size
s-s SDS, heat
Proteins with SDS
+
–
How Does an SDS-PAGE Gel Work?
Negatively charged proteins move to positive electrode
Smaller proteins move faster
Proteins separate by size
+
s-s SDS, heat
Proteins with SDS
–
SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) SDS Detergent (Sodium
Dodecyl Sulfate) Solubilizes and denatures
proteins Adds negative charge to
proteins
Heat denatures proteins
O S
O
O
O
-
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH3
SDS
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 DaAverage nucleotide pair = 649 Da1 kilobase of DNA = 650 kDa1 kilobase of DNA encodes 333 amino acids = 36 kDa
Gel Analysis
Lane1. Kaleidoscope Markers2. Shark3. Salmon4. Trout5. Catfish6. Sturgeon7. Actin and Myosin
Standard
Molecular Weight Estimation
0
50
100
150
200
250
0 20 40 60
Distance (mm from well)
Siz
e in
kD
a
kDa mm203 8.5135 12.086 18.5
41 28.0
33 34.0
19 41.5
8 44.5
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