Exploration of Phylogeny of
Serum Proteins Using the
Western Press-Blot Procedure
Introduction
1.
2.
3.
4.
5.
Studying molecular evolution
Types of antibodies
Antibodies and relatedness
Laboratory results
Conclusion
Section I:
Studying Molecular Evolution
Overview: Studying Molecular
Evolution
• Two ways to study evolutionary
relationships:
–Protein sequence analysis
–Immunologic cross reactivity
Protein Sequence Analysis
• Comparison of proteins in
different organisms (ex.
Cytochrome C, DNA)
• Useful in research
Studying Molecular Evolution
• Two ways to study evolutionary
relationships:
–Protein sequence analysis
–Immunologic cross reactivity
Section II:
Immunologic Cross Reactivity
(Antibodies and Relatedness)
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?
Overview: Antibodies and
Relatedness
4. What does this tell us about
evolutionary relatedness?
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?
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
Antibodies and Relatedness
• Antigens
• macromolecules that elicit an
immune response in the body
• most commonly composed of
proteins or polysaccharides
Antibodies and Relatedness
• Epitope
–also called an antigenic
determinant
– the specific portion of antigen to
which the antibody binds
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
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?
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?
Antibodies and Relatedness
3. What does this tell us about
evolutionary relatedness?
Section III:
Types of Antibodies
Overview: Types of Antibodies
1. What are monoclonal and
polyclonal antibodies?
2. Why does this matter?
(Examples of antibodies in
medicine)
Monoclonal and Polyclonal
Antibodies
• Monoclonal antibodies
– homogeneous
– synthesized from a population of
identical cells (clones)
– antibodies all target the same
epitope on an antigen
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
Overview: Types of Antibodies
1. What are monoclonal and
polyclonal antibodies?
2. Why does this matter?
(Examples of antibodies in
medicine)
Why does this matter?
• We used polyclonal antibodies
in our experiment
• knowing the difference is
important – why?
Antibodies and Medicine
• Vitaxin
–medicine which uses monoclonal
antibodies
–binds to a specific vascular
integrin found on blood vessels of
tumors
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
Section IV:
Laboratory Results
Method
Electrophoresis
of gel
Preparation
of Western
Blot
Primary
Antibody
Reaction
Stain of
Nitrocellulose
Blot
Color
Development
Preparation of Nitrocelluose
Membrane
Nitrocellulose
membrane
Separated Proteins
Gelatin
Primary antibody
Secondary antibody
(Color Development
Solution)
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
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
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
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
Derived Phylogenetic Relationships
Closely Related Somewhat Related Distantly Related
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