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Bis 104 Lecture 3

Bis 104 Lecture 3 - Bis 104 Lecture 3 INDIRECT...

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Bis 104 Lecture 3 INDIRECT IMMUNOFLUORESCENE MICROSCOPY Primary antibodies: rabbit antibodies directed against antigen A Secondary antibodies: marker-coupled antibodies directed against rabbit antibodies This process amplify signal. This allows you to visualize antigen very clearly. Visualizing different components in a mitotic cell: Spindle microtubules: anti-tubulin antibodies Kinetochores: anti-CENP antibodies DNA: dapi FLUORESCENT DYE IMMUNOGOLD ELECTRON MICROSCOPY Insulin molecules are detected by EM using anti-unsulin antibodies bound to tiny gold particles (10- 50nm) Q: Why did gold particles appear in black under TEM? A: Because the light doesn’t pass through. Antibody column affinity chromatography Using antibodies to purify molecules Add specific antibodies to molecules By going through immunoaffinity column chromatography, you can separate which molecule you want. Detecting interaction among proteins I. IMMUNOPRECIPITATION Can separate protein by binding antibodies to proteins, denature via boiling, than going through the SDS- PAGE. II. YEAST TWO-HYBRID ASSAY Gal4 transcription factor from yeast is modified by recombinant DNA technology. III. PROTEIN COMPLEX PUURIFICATION AIDED BY AFFINITY TAGS
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Bis 104 Lecture 3 - Bis 104 Lecture 3 INDIRECT...

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