Immunoprecipitation of mRNA-protein complexes.
Immunoprecipitation of mRNA-protein complexes is a method that can be used to study RNA binding protein (RBP)-RNA interactions. In this protocol, an antibody targeting an RBP of interest is used to immunoprecipitate the RBP and any interacting molecules from a cell lysate. Reverse transcription followed by PCR is then used to identify individual mRNAs isolated with the RBP. This method focuses on examining an association between a specific RBP-mRNA complex, and it is best suited for a small scale screening of known or putative binding partners. It can also be used as a second, independent method to verify RBP-mRNA interactions discovered through more universal screening techniques. We describe the immunoprecipitation protocol in practical detail and discuss variations of the method as well as issues associated with it. The procedure takes three days to complete.
Peritz
Tiina
T
Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
Zeng
Fanyi
F
Kannanayakal
Theresa J
TJ
Kilk
Kalle
K
Eiríksdóttir
Emelía
E
Langel
Ulo
U
Eberwine
James
J
D000818:Animals; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D047468:Immunoprecipitation / Q000379:methods*; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry* / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D051381:Rats
D012333:RNA, Messenger; D016601:RNA-Binding Proteins