Immunopurification and analysis of protein and RNA components of mRNP in mammalian cells.
We describe a basic, fast, and reliable technique to isolate and characterize ribonucleoprotein (RNP) using antibody to a constituent protein. The antibody serves to immunopurify RNP from total cells or nuclear and cytoplasmic cell fractions under conditions that promote RNP integrity. The presence of other RNP proteins as well as transcripts can then be analyzed by Western blotting and reverse transcription polymerase chain reaction, respectively. RNase treatment before immunopurification can be used to assess the dependence of protein-protein interactions on RNA. We also describe a modification using beta-mercaptoethanol that facilitates analyzing proteins that comigrate with antibody light or heavy chains.
Lejeune
Fabrice
F
Department of Biochemistry and Biophysics, School of Medicine and Dentistry, University of Rochester, NY, USA.
Maquat
Lynne E
LE
D000818:Animals; D015153:Blotting, Western / Q000379:methods; D019556:COS Cells; D002458:Cell Fractionation / Q000379:methods*; D002467:Cell Nucleus; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D003593:Cytoplasm; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000276:immunology / Q000302:isolation & purification; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology / Q000378:metabolism*; D008623:Mercaptoethanol / Q000378:metabolism; D039502:Nuclear Cap-Binding Protein Complex / Q000276:immunology / Q000302:isolation & purification; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012260:Ribonucleases / Q000494:pharmacology; D012261:Ribonucleoproteins / Q000276:immunology / Q000302:isolation & purification*; D014157:Transcription Factors / Q000276:immunology / Q000302:isolation & purification
D007074:Immunoglobulin G; D039502:Nuclear Cap-Binding Protein Complex; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D014157:Transcription Factors; C468286:UPF2 protein, human; C435487:UPF3A protein, human; C026452:messenger ribonucleoprotein; D008623:Mercaptoethanol; D012260:Ribonucleases; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase