Protocol

mRNA Processing and Metabolism

Volume 257 of the series Methods in Molecular Biology™ pp 115-124

Immunopurification and Analysis of Protein and RNA Components of mRNP in Mammalian Cells

  • Fabrice LejeuneAffiliated withDepartment of Biochemistry and Biophysics, School of Medicine and Dentistry, University of Rochester
  • , Lynne E. MaquatAffiliated withDepartment of Biochemistry and Biophysics, School of Medicine and Dentistry, University of Rochester

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Abstract

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 β-mercaptoethanol that facilitates analyzing proteins that comigrate with antibody light or heavy chains.

Key Words

Immunopurification mRNP Western blotting RT-PCR RNase treatment β-mercapto-ethanol