Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Typical IP Protocol
Document related concepts
Rosetta@home wikipedia , lookup
Protein design wikipedia , lookup
Circular dichroism wikipedia , lookup
Homology modeling wikipedia , lookup
Protein domain wikipedia , lookup
Protein folding wikipedia , lookup
Bimolecular fluorescence complementation wikipedia , lookup
Protein structure prediction wikipedia , lookup
List of types of proteins wikipedia , lookup
Protein moonlighting wikipedia , lookup
Immunoprecipitation wikipedia , lookup
Intrinsically disordered proteins wikipedia , lookup
Gel electrophoresis wikipedia , lookup
Nuclear magnetic resonance spectroscopy of proteins wikipedia , lookup
Protein mass spectrometry wikipedia , lookup
Protein–protein interaction wikipedia , lookup
Transcript
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 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 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 agarose G 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, posttranslational events, etc. Immunoblotting • • aka Western Blotting use Ab to detect protein after electrophoresis 1. 2. 3. 4. 5. Protein electrophoresis Transfer proteins to membrane Incubate membrane with antibody Extensive washing 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 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 Immunoassay Uses • antigen detection, characterization, (quantification?) • antibody detection, characterization, (quantification?)
