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Antibodies Analytical Techniques Utilizing Antibodies: flow cytometry

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Presentation on theme: "Antibodies Analytical Techniques Utilizing Antibodies: flow cytometry"— Presentation transcript:

1 Antibodies Analytical Techniques Utilizing Antibodies: flow cytometry
gel electrophoresis immunoprecipitation (IP) immunoblotting microscopy immunofluorescence (IFA) electron microscopy ELISA CNBr 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 Typical IP Protocol G agarose 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 agarose 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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