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Analytical Techniques Utilizing Antibodies : flow cytometry gel electrophoresis

CNBr. . Antibodies. Analytical Techniques Utilizing Antibodies : flow cytometry gel electrophoresis immunoprecipitation (IP) immunoblotting microscopy immunofluorescence (IFA) electron microscopy ELISA. antibodies bind proteins with high specificity and affinity

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Analytical Techniques Utilizing Antibodies : flow cytometry gel electrophoresis

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  1. CNBr  Antibodies • Analytical Techniques • Utilizing Antibodies: • flow cytometry • gel electrophoresis • immunoprecipitation (IP) • immunoblotting • microscopy • immunofluorescence (IFA) • electron microscopy • ELISA • antibodies bind proteins with high specificity and affinity • affinity chromatography • analytical techniques

  2. Immunoprecipitation • affinity purification based on isolation of Ag-Ab complexes • analyze by gel electrophoresis • initially based on centrifugation of large supramolecular complexes • [high] and equal amounts • isolation of Ag-Ab complexes • fixed S. aureus • protein A-agarose • protein G-agarose • Bacterial proteins that bind IgG (Fc): • protein A (Staphylococcus aureus) • protein G (Streptococcus) • binds more species and subclasses

  3. agarose Typical IP Protocol • 1. Solubilize antigen • usually non-denaturing • SDS + excess of TX100 • 2. Mix extract and Ab • 3. Add protein G-agarose, etc • 4. Extensively wash • 5. Elute with sample buffer • 6. SDS-PAGE • 7. Detection • protein stain • radioactivity G

  4. Radiolabeling of Proteins • carried out before IP • metabolic (amino acids or other precursors + cells) • chemically (eg., iodination) • IP and SDS-PAGE • detect by autoradiography or fluorography following electrophoresis • also provides information about synthesis, post-translational events, etc.

  5. Immunoblotting • aka Western Blotting • use Ab to detect protein after electrophoresis • 1. Protein electrophoresis • 2. Transfer proteins to membrane • 3. Incubate membrane with antibody • 4. Extensive washing • 5. Detect bound antibody • - radioactive 2nd Ab or protein A • - ELISA

  6. Enzyme-Linked ImmunoSorbent Assay

  7. Detection by Chemiluminescence

  8. Western Blot vs Immunoprecipitation • Experimental Design • eg., synthesis (IP) • Ag concentration • IP better for low abundance proteins • Ag solubility • Western for insoluble proteins • Ab recognition • conformational dependent epitopes • 4o structure

  9. Combined Immunoprecipitation and Immunoblotting • carry out IP and electrophoresis • transfer to membrane • use same or different Ab against Ag in blotting • need ‘no extract’ control

  10. Immunoassay Uses • antigen detection, characterization, (quantification?) • antibody detection, characterization, (quantification?)

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