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Analytical Techniques Utilizing Antibodies: flow cytometry gel electrophoresis immunoprecipitation (IP) immunoblotting microscopy immunofluorescence (IFA)

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Presentation on theme: "Analytical Techniques Utilizing Antibodies: flow cytometry gel electrophoresis immunoprecipitation (IP) immunoblotting microscopy immunofluorescence (IFA)"— Presentation transcript:

1 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 Antibodies CNBr

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 Typical IP Protocol 1. Solubilize antigen usually non-denaturing SDS + excess of TX 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 agarose

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 2 nd 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 4 o structure

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

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


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