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

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

Introduction

• 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

• Definitions and Background

• How do you produce anti-human albumin antibodies?

• Why do these anti-human albumin antibodies interact with serum albumin from animals other than humans?

Overview: Antibodies and Relatedness

• What does this tell us about evolutionary relatedness?

Antibodies and Relatedness

• Definitions and Background

• How do you produce anti-human albumin antibodies?

• 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

• Definitions and Background

• How do you produce anti-human albumin antibodies?

• Why do these anti-human albumin antibodies interact with serum albumin from animals other than humans?

Antibodies and Relatedness

• Definitions and Background

• How do you produce anti-human albumin antibodies?

• Why do these anti-human albumin antibodies interact with serum albumin from animals other than humans?

Antibodies and Relatedness

• What does this tell us about evolutionary relatedness?

Section III: Types of Antibodies

Overview: Types of Antibodies

• What are monoclonal and polyclonal antibodies?

• 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

• What are monoclonal and polyclonal antibodies?

• 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

Preparation of Nitrocelluose Membrane

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

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