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

Introduction 1.Studying molecular evolution 2.Types of antibodies 3.Antibodies and relatedness 4.Laboratory results 5.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 Stain of Nitrocellulose Blot Primary Antibody Reaction Color Development

Preparation of Nitrocelluose Membrane Nitrocellulose membrane Separated 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 GlobulinsBovine Serum, Goat Serum, and Sheep Serum are closely related to Bovine Albumin and Bovine Gamma Globulins Horse Serum is somewhat related to the antibodiesHorse Serum is somewhat related to the antibodies Chicken Serum and transferrin may be distantly related to the antibodiesChicken Serum and transferrin may be distantly related to the antibodies

Derived Phylogenetic Relationships Closely RelatedSomewhat RelatedDistantly Related

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