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Analyze protein profiles from fresh muscle tissue by… Using polyacrylamide electrophoresis to separate proteins by size Examine phylogenetic relationships based on muscle protein profiles. Traditional classification based upon traits, especially morphological characters.

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Presentation Transcript
slide1
Analyze protein profiles from fresh muscle tissue by…
  • Using polyacrylamide electrophoresis to separate proteins by size
  • Examine phylogenetic relationships based on muscle protein profiles
biochemical similarities

DNA

RNA

Protein

Trait

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
slide4
Experiment: Compare protein profiles various fresh muscle tissue.
  • Procedure:
    • Extract proteins from tissue
    • Denature proteins
    • Separate proteins by size using polyacrylamide gel electrophoresis
    • Stain to visualize protein bands
    • Analyze and interpret gels
protein size comparison
Protein size comparison
  • Break protein complexes into individual proteins
  • Denature proteins using detergent and heat
  • Separate proteins based on size
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

Why use polyacrylamide gels to separate proteins?
protein size
Protein size
  • Size measured in kilodaltons (kDa)
  • Dalton = mass of hydrogen atom

= 1.66 x 10 -24 gram

  • Average amino acid = 110 daltons
muscle contains proteins of many sizes
Muscle contains proteins of many sizes

ProteinkDaFunction

titin 3000 center myosin in sarcomere

dystrophin 400 anchoring to plasma membrane

filamin 270 cross-link filaments into gel

myosin heavy chain 210slide filaments

spectrin 265 attach filaments to plasma membrane

nebulin 107 regulate actin assembly

a-actinin 100 bundle filaments

gelosin 90 fragment filaments

fimbrin 68 bundle filaments

actin42form filaments

tropomyosin 35 strengthen filaments

myosin light chain 27slide 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….

actin and myosin
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
what s in the sample buffer
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
why heat the samples
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, ß-Me,

heat

-

+

proteins with

SDS

How does an SDS-PAGE gel work?
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

Gel Analysis
molecular weight analysis
kDa mmMolecular Weight Analysis

203 8.5

135 12.0

86 18.5

41 28.0

33 34.0

19 41.5

8 44.5

phylogenetic tree
Phylogenetic Tree

TUNA

MACKEREL

SALMON

TROUT

CARP

MINNOW

SNAPPER

PERCH

WALLEYE

BASS

CATFISH

COD

HAKE

POLLOCK

SMELT

ANCHOVIES HERRINGS SARDINES

FLOUNDER

SOLE

HALIBUT

PIKE

STURGEON

GAR

SHARK

Agnatha

Chondrichthyes

Ostheichthyes

Amphibia

Reptilia

Aves

Mammalia

OYSTER

CLAM

MUSSEL

SCALLOP

OCTOPUS

SQUID

CRAB

LOBSTER

SHRIMP

Mollusk

Arthropod

Echinoderm

Chordate

Deuterostome

Protostome

Metazoa