Exploration of Phylogeny of Serum Proteins Using the

Western Press-Blot Procedure

Introduction

1. Studying molecular evolution2. Types of antibodies3. Antibodies and relatedness4. Laboratory results5. 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 Background2. 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 Background2. 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 Background2. 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 Background2. 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

Stain of Nitrocellulose

Blot

Primary Antibody Reaction

Color Development

Preparation of Nitrocelluose Membrane

Nitrocellulose membrane

SeparatedSeparated Proteins

GelatinGelatin

Primary antibodyPrimary antibody

Secondary antibody Secondary antibody (Color Development (Color Development Solution)Solution)

Stained Nitrocellulose Blot

1: Bovine Gamma 1: Bovine Gamma GlobulinGlobulin

2: BSA2: BSA3: Bovine Transferrin3: Bovine Transferrin4: Bovine Serum4: Bovine Serum5: Goat Serum5: Goat Serum6: Sheep Serum6: Sheep Serum7: Horse Serum7: Horse Serum8: Chicken Serum8: Chicken Serum

Results for Bovine Serum Albumin

1: Bovine Gamma 1: Bovine Gamma GlobulinGlobulin

2: BSA2: BSA3: Bovine Transferrin3: Bovine Transferrin4: Bovine Serum4: Bovine Serum5: Goat Serum5: Goat Serum6: Sheep Serum6: Sheep Serum7: Horse Serum7: Horse Serum8: Chicken Serum8: Chicken Serum

Results for Bovine Gamma Globulin

1: Bovine Gamma 1: Bovine Gamma GlobulinGlobulin

2: BSA2: BSA3: Bovine Transferrin3: Bovine Transferrin4: Bovine Serum4: Bovine Serum5: Goat Serum5: Goat Serum6: Sheep Serum6: Sheep Serum7: Horse Serum7: Horse Serum8: Chicken Serum8: Chicken Serum

Conclusion

• Bovine Serum, Goat Serum, and Sheep Bovine Serum, Goat Serum, and Sheep Serum are closely related to Bovine Serum are closely related to Bovine Albumin and Bovine Gamma GlobulinsAlbumin and Bovine Gamma Globulins

• Horse Serum is somewhat related to the Horse Serum is somewhat related to the antibodiesantibodies

• Chicken Serum and transferrin may be Chicken Serum and transferrin may be distantly related to the antibodiesdistantly related to the antibodies

Derived Phylogenetic Relationships

Closely Related Somewhat Related Distantly Related

Summary

• Antigen/antibody relationship useful in Antigen/antibody relationship useful in studying evolutionstudying evolution

• Western blotting used to demonstrate Western blotting used to demonstrate phylogenetic relationshipsphylogenetic relationships

• Phylogenies help us explore possibilities Phylogenies help us explore possibilities of other related characteristicsof other related characteristics

• Findings are applied medicine & scientific Findings are applied medicine & scientific researchresearch