Exploration of Phylogeny of Serum Proteins Using the Western Press-Blot Procedure

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Exploration of Phylogeny of Serum Proteins Using
the Western Press-Blot Procedure
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Introduction

1. Studying molecular evolution
2. Types of antibodies
3. Antibodies and relatedness
4. Laboratory results
5. Conclusion

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Section IStudying Molecular Evolution
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Overview Studying Molecular Evolution

• Two ways to study evolutionary relationships
• Protein sequence analysis
• Immunologic cross reactivity

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Protein Sequence Analysis

• Comparison of proteins in different organisms
  (ex. Cytochrome C, DNA)
• Useful in research

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Studying Molecular Evolution

• Two ways to study evolutionary relationships
• Protein sequence analysis
• Immunologic cross reactivity

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Section IIImmunologic Cross Reactivity(Antibod
ies and Relatedness)
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Overview Antibodies and Relatedness

1. Definitions and Background
2. How do you produce anti-human albumin antibodies?
3. Why do these anti-human albumin antibodies
   interact with serum albumin from animals other
   than humans?

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Overview Antibodies and Relatedness

1. What does this tell us about
   evolutionary relatedness?

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Antibodies and Relatedness

1. Definitions and Background
2. How do you produce anti-human albumin antibodies?
3. Why do these anti-human albumin antibodies
   interact with serum albumin from animals other
   than humans?

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Antibodies and Relatedness

• Antibodies
• glycoproteins made of two subunits
• perform two functions
• recognize and bind to an epitope on an antigen
• trigger a useful response to an antigen

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Antibodies and Relatedness

• Antigens
• macromolecules that elicit an immune response in
  the body
• most commonly composed of proteins or
  polysaccharides

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Antibodies and Relatedness

• Epitope
• also called an antigenic determinant
• the specific portion of antigen to which the
  antibody binds

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Antibodies and Relatedness

• Blood Serum
• the clear liquid that separates from blood when
  clotting occurs
• Serum Albumin
• the major plasma protein
• carries large inorganic anions and hormones

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Antibodies and Relatedness

1. Definitions and Background
2. How do you produce anti-human albumin antibodies?
3. Why do these anti-human albumin antibodies
   interact with serum albumin from animals other
   than humans?

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Antibodies and Relatedness

1. Definitions and Background
2. How do you produce anti-human albumin antibodies?
3. Why do these anti-human albumin antibodies
   interact with serum albumin from animals other
   than humans?

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Antibodies and Relatedness

1. What does this tell us about
   evolutionary relatedness?

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Section IIITypes of Antibodies
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Overview Types of Antibodies

• What are monoclonal and polyclonal antibodies?
• Why does this matter? (Examples of antibodies in
  medicine)

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Monoclonal and Polyclonal Antibodies

• Monoclonal antibodies
• homogeneous
• synthesized from a population of identical cells
  (clones)
• antibodies all target the same epitope on an
  antigen

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Monoclonal and Polyclonal Antibodies

• Polyclonal antibodies
• Heterogenous
• Mixture of several types of antibodies to many
  different epitopes on the same protein antigen
• The typical response to an antigen in eukaryotes

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Overview Types of Antibodies

• What are monoclonal and polyclonal antibodies?
• Why does this matter? (Examples of antibodies in
  medicine)

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Why does this matter?

• We used polyclonal antibodies in our experiment
• knowing the difference is important why?

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Antibodies and Medicine

• Vitaxin
• medicine which uses monoclonal antibodies
• binds to a specific vascular integrin found on
  blood vessels of tumors

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Antibodies and Medicine

• Vitaxin
• this specificity means Vitaxin will not bind to
  blood vessels in healthy tissue
• polyclonal antibodies used this way would kill
  cancer and healthy cells by attaching to common
  epitopes

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Section IVLaboratory Results
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Method
Electrophoresis of gel
Preparation of Western Blot
Stain of Nitrocellulose Blot
Primary Antibody Reaction
Color Development
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Preparation of Nitrocelluose Membrane
Nitrocellulose membrane
Separated Proteins
Gelatin
Primary antibody
Secondary antibody (Color Development Solution)
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Stained Nitrocellulose Blot

• 1 Bovine Gamma Globulin
• 2 BSA
• 3 Bovine Transferrin
• 4 Bovine Serum
• 5 Goat Serum
• 6 Sheep Serum
• 7 Horse Serum
• 8 Chicken Serum

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Results for Bovine Serum Albumin

• 1 Bovine Gamma Globulin
• 2 BSA
• 3 Bovine Transferrin
• 4 Bovine Serum
• 5 Goat Serum
• 6 Sheep Serum
• 7 Horse Serum
• 8 Chicken Serum

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Results for Bovine Gamma Globulin

• 1 Bovine Gamma Globulin
• 2 BSA
• 3 Bovine Transferrin
• 4 Bovine Serum
• 5 Goat Serum
• 6 Sheep Serum
• 7 Horse Serum
• 8 Chicken Serum

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Conclusion

• Bovine Serum, Goat Serum, and Sheep Serum are
  closely related to Bovine Albumin and Bovine
  Gamma Globulins
• Horse Serum is somewhat related to the antibodies
• Chicken Serum and transferrin may be distantly
  related to the antibodies

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Derived Phylogenetic Relationships
Closely Related
Somewhat Related
Distantly Related
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Summary

• Antigen/antibody relationship useful in studying
  evolution
• Western blotting used to demonstrate phylogenetic
  relationships
• Phylogenies help us explore possibilities of
  other related characteristics
• Findings are applied medicine scientific
  research

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