1 Antibodies Analytical Techniques Utilizing Antibodies: flow cytometry gel electrophoresisimmunoprecipitation (IP)immunoblottingmicroscopyimmunofluorescence (IFA)electron microscopyELISACNBrantibodies bind proteins with high specificity and affinityaffinity chromatographyanalytical techniques
2 Immunoprecipitationaffinity purification based on isolation of Ag-Ab complexesanalyze by gel electrophoresisinitially based on centrifugation of large supramolecular complexes[high] and equal amountsisolation of Ag-Ab complexesfixed S. aureusprotein A-agaroseprotein G-agaroseBacterial 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-denaturingSDS + excess of TX1002. Mix extract and Ab3. Add protein G-agarose, etc4. Extensively wash5. Elute with sample buffer6. SDS-PAGE7. Detectionprotein stainradioactivityagaroseG
4 Radiolabeling of Proteins carried out before IPmetabolic (amino acids or other precursors + cells)chemically (eg., iodination)IP and SDS-PAGEdetect by autoradiography or fluorography following electrophoresisalso provides information about synthesis, post-translational events, etc.
5 Immunoblotting aka Western Blotting use Ab to detect protein after electrophoresis1. Protein electrophoresis2. Transfer proteins to membrane3. Incubate membrane with antibody4. Extensive washing5. Detect bound antibody- radioactive 2nd Ab or protein A- ELISA
8 Western Blot vs Immunoprecipitation Experimental Designeg., synthesis (IP)Ag concentrationIP better for low abundance proteinsAg solubilityWestern for insoluble proteinsAb recognitionconformational dependent epitopes4o structure
9 Combined Immunoprecipitation and Immunoblotting carry out IP and electrophoresistransfer to membraneuse same or different Ab against Ag in blottingneed ‘no extract’ control