Transcript Are All Fish Related? A look at biochemical evidence for

• Analyze protein profiles
from fresh muscle tissue
by…
• Using polyacrylamide
electrophoresis to
separate proteins by size
• Examine phylogenetic
relationships based on
muscle protein profiles
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Traditional Systematics and
Taxonomy
• Traditional classification based upon
traits, especially morphological
characters.
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Biochemical Similarities
• Traits are discrete characteristics. In biology we are
concerned with “heritable traits.”
• Proteins are generally responsible for traits, most
often as enzymes affecting biochemical pathways or
as structural or dynamic elements of the cytoskeleton.
• DNA codes for proteins that confer traits
DNA
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RNA
Protein Trait
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• Experiment: Compare protein profiles various
fresh muscle tissue.
• Procedure:
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–
–
–
–
Extract proteins from tissue
Denature proteins
Separate proteins by size using polyacrylamide gel
electrophoresis
Stain to visualize protein bands
Analyze and interpret gels
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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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Why use polyacrylamide
gels to separate proteins?
• Acrylamide gel
tight matrix
• Ideal for protein separation
• Smaller pore size than agarose
• Proteins much smaller than DNA
– average amino acid = 110 Da
– average base pair = 649 Da
– 1 kilobase of DNA = 650 kDa
– 1 kilobase of DNA encodes 333 amino acids = 36 kDa
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Protein size
• Size measured in kilodaltons (kDa)
• Dalton = mass of hydrogen atom
= 1.66 x 10 -24 gram
• Average amino acid = 110 daltons
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Muscle contains proteins of
many sizes
Protein
kDa
Function
titin
dystrophin
filamin
3000
400
270
center myosin in sarcomere
anchoring to plasma membrane
cross-link filaments into gel
myosin heavy chain
210
slide filaments
spectrin
nebulin
a-actinin
gelosin
fimbrin
265
107
100
90
68
attach filaments to plasma membrane
regulate actin assembly
bundle filaments
fragment filaments
bundle filaments
actin
42
form filaments
tropomyosin
35
strengthen filaments
myosin light chain
27
slide filaments
troponin (T, I, C)
30, 19, 17
mediate regulation of contraction
thymosin
5
sequester actin monomers
These are generalized molecular masses and may vary in different species….
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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 of two heavy subunits
(220 kDa)
and two light subunits (20 kDa)
• Breaks down ATP for muscle
contraction
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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
• Bromphenol Blue dye to visualize
samples
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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
+
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How does an SDS-PAGE gel
work?
• Negatively charged proteins
move to positive electrode
s-s
• Smaller proteins move faster
• Proteins separate by size
SDS, ß-Me,
heat
proteins with
SDS
-
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+
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Gel Analysis
15% SDS-PAGE
• Lane 1: Marker
• Lane 2: Shark
• Lane 3: Salmon
• Lane 4: Rainbow trout
• Lane 5: Cod
• Lane 6: Sturgeon
• Lane 8: Croaker
• Lane 9: Weakfish
• Lane 10: Actin/myosin
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Molecular Weight Analysis
mm
203
135
86
8.5
12.0
18.5
41
28.0
33
34.0
250
200
kDa
kDa
150
100
50
0
0
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41.5
8
44.5
20
40
60
mm from well
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Phylogenetic Tree
SALMON
TROUT
CARP
MINNOW
CATFISH
SMELT
ANCHOVIES
HERRINGS
SARDINES
PIKE
COD
HAKE
POLLOCK
SNAPPER
PERCH
WALLEYE
BASS
TUNA
MACKEREL
FLOUNDER
SOLE
HALIBUT
STURGEON
GAR
SHARK
Agnatha
Chondrichthyes
OYSTER
CLAM
MUSSEL
SCALLOP
OCTOPUS
SQUID
CRAB
LOBSTER
SHRIMP
Mollusk
Arthropod
Protostome
Ostheichthyes
Echinoderm
Amphibia
Reptilia
Aves
Mammalia
Chordate
Deuterostome
Metazoa
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