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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
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
Typical IP Protocol1. Solubilize antigen• usually non-denaturing• SDS + excess of TX100
2. Mix extract and Ab 3. Add protein G-agarose, etc4. Extensively wash5. Elute with sample buffer6. SDS-PAGE7. Detection• protein stain• radioactivity
Gagarose
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.
Immunoblotting• aka Western Blotting• use Ab to detect protein after
electrophoresis
1. Protein electrophoresis2. Transfer proteins to membrane3. Incubate membrane with antibody4. Extensive washing5. Detect bound antibody
- radioactive 2nd Ab or protein A - ELISA
Enzyme-Linked ImmunoSorbent Assay
Detection by Chemiluminescence
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
• 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
• antigen detection, characterization, (quantification?)• antibody detection, characterization, (quantification?)
Immunoassay Uses