BE115+-+Lecture07 - Projects Form a project group (4...

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Projects Form a project group (4 people) and be ready to suggest and discuss a general idea for a project to me during this week’s lab Concrete goals to be finalized by Week 9 Weblog post due by 5PM on 11/1, no exceptions Project presentations: Thursday, 12/9, Noon-3 Project paper due: Thursday, 12/9 at noon 1
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Lecture 7 Immunological Methods in Tissue Engineering “PHYSICIAN, n. One upon whom we set our hopes when ill and our dogs when well.” - Ambrose Bierce 2
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3 Nitrocellulose membrane Developed blot with Abs for red protein Gel Commassie Gel
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Western Blot 4 SDS-PAGE gel Nitrocellulose membrane
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Immunology Behring and Kitasato discovered that animals infected with tetanus and diphtheria had something protective in their blood serum which unvaccinated animals did not The blood serum could also protect unvaccinated animals Erlich suggested that the serum contained proteins that he called “antibodies”, which fit disease antigens “like a lock and key” 5
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Antibodies Antibodies stick to (recognize) other molecules ~ 20% of serum proteins are antibodies Five types: IgA (~10-15%), IgD, IgM (~5-10%), IgE, IgG (~80%) Each type has specialized function and there are various subclasses of each type IgG is most commonly used in experiments 6
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Antibodies Y-shaped proteins of ~ 150,000 MW The two upper ends of the Y are where binding occurs The lower end of the Y contains the effector region 7
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Nomenclature Antigen – the molecule that an antibody recognizes (often a protein) Epitope – the specific part of the antigen recognized by the antibody Source – the animal from which the antibody came from 8
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Molecular Immunology Antibodies stick because of reversible non- covalent interactions between antibody and antigen Note that the antibody shown here recognizes a small part (the epitope) of a large molecule (the 9 Antibody (grey/red/blue) Antigen (egg white lysozyme, green) Water (blue)
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Immunostaining: An Act in Three Parts 10
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Immunostaining Basics 11 Three distinct proteins Add a labeled secondary antibody which recognizes the primary antibody * * * * * * * * * * * * * * * * * * * * * * * * * A surface coated with proteins A slide with fixed cells A nitrocellulose membrane after SDS-PAGE and transfer * * * * * * * * * * * * * * * * Add primary antibody which recognizes
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Primary …is produced in small amounts …is expensive …can be relatively fragile Secondary… …is produced in large amounts …is cheap In addition, more than one secondary antibody can bind to a single primary antibody, which boosts the signal! 12
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This note was uploaded on 10/14/2011 for the course BIOE 115 taught by Professor Haley during the Spring '11 term at City College of San Francisco.

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BE115+-+Lecture07 - Projects Form a project group (4...

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