Optimization of immunoprecipitation-western blot analysis in detecting GW182-associated components of GW/P bodies.
Characterizing the components of GW/processing bodies is key to elucidating RNA interference and messenger RNA processing pathways. This protocol addresses challenges in isolating a low-abundance protein GW182 and GW body (GWB)-associated proteins by building on previous reports that used polyclonal sera containing autoantibodies to GW/P body components. This protocol uses commercially available monoclonal antibodies to GW182 that are covalently coupled to Protein A or G sepharose beads and then used to immunoprecipitate GW182 and associated proteins from cell extracts. Immunoprecipitates are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes and probed by western blot with antibodies directed to proteins of interest. This protocol, which is expected to take 4-5 d, provides a biochemical approach for detecting GW182 and associated proteins in biological samples and thus facilitates the elucidation of the diverse functions of GWBs. It is expected that this protocol can be adapted to the detection of other RNA-binding complexes.
Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada.
Joanna J
JJ
Moser
Edward K L
EK
Chan
Marvin J
MJ
Fritzler
D060565:Argonaute Proteins; D001324:Autoantigens / Q000032:analysis* / Q000378:metabolism; D015153:Blotting, Western / Q000379:methods*; D002268:Carboxypeptidases / Q000032:analysis / Q000737:chemistry; D003101:Collodion / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism; D005095:Exoribonucleases / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods*; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins; D043244:Ribonuclease III / Q000378:metabolism; D012685:Sepharose
C466422:AGO2 protein, human; D060565:Argonaute Proteins; D001324:Autoantigens; D015852:Eukaryotic Initiation Factor-2; D008869:Microtubule-Associated Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C465663:TNRC6A protein, human; D003101:Collodion; D012685:Sepharose; D005095:Exoribonucleases; C470512:XRN1 protein, human; D043244:Ribonuclease III; D002268:Carboxypeptidases; C436327:SCPEP1 protein, human