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RNP-IP (Original Method): Obtaining Majority RNA from RNA Binding Protein in the Nucleus
Published: May 20, 2012 DOI: 10.21769/BioProtoc.218 Views: 19210
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Abstract
Post-transcriptional regulation of gene expression is a ribonucleoprotein (RNP)-driven process, which involves RNA binding proteins (RBPs) and noncoding RNAs that regulate splicing, nuclear export, subcellular localization, mRNA stability and translation. mRNAs encoding proteins that function in a particular cell process or pathway can be found within a unique mRNP complex, which consists of mRNA and RNP. This provides valuable information regarding not only known components of a particular process or pathway, but importantly, leads to the identification of novel components representing potential therapeutic targets and biomarkers. In addition to those targets identified by pathway expansion, the specific RBPs (RNA binding proteina) regulating RNA functions may be potential therapeutic targets in their own right. RNP-IP is a technology that allows the isolation and identification of mRNAs, microRNAs and protein components of RNP complexes from cell extracts using antibodies to RBPs. Once purified, the RNAs present in the complex are analyzed to identify the target mRNAs using various molecular biology tools such as RT-PCR, gene expression analysis based on microarray technology (Chip analysis), or sequencing. Using this method, more RNA that is present in the nucleus can be obtained.
Materials and Reagents
- Normal Rabbit IgG
- High-salt solution
- RIP-certified antibody (NBL, catalog number depends on what do you want to target)
- Protease inhibitor (Molecular Biology)
- Aprotinin
- Leupeptin
- Phenylmethylsulfonyl fluoride (PMSF)
- RNase inhibitor (Life Technologies, Invitrogen™, catalog number: 10777-019 )
- Dithiothreitol (DTT) (reducing agent)
- Protein A beads (GE Healthcare Dharmacon, catalog number: 17-0780-01 ) or Protein G beads (Thermo Fisher Scientific, catalog number: 22852 )
- Ethanol (Molecular Biology)
- 2-Propanol (Molecular Biology)
- Nuclease-free PBS
- Isotype control IgG (if necessary)
- Kit components (see Recipes)
- Commercial reagent (see Recipes)
- Buffer preparation (see Recipes)
- Wash buffer (see Recipes)
- Precaution: Additional buffer preparation (see Recipes)
- Preparation of antibody-immobilized protein A or protein G agarose beads-RNP complex
Equipment
- Microcentrifuge capable of 15,000 x g
- Microcentrifuge tube (1.5 ml or 2 ml) (Nuclease-free) (recommendation; use low-adhesion tube for RIP-Assay)
- Centrifuge capable of 2,000 x g
- Centrifuge tube (15 ml or 50 ml)
- Pipette (5 ml, 10 ml, 25 ml) (nuclease-free)
- Pipette tip (10 μl, 20-100 μl , 200 μl , and 1,000 μl) (nuclease-free) (recommendation; use low-adhesion pipette tip for RIP-Assay)
- Ultra low temperature freezer (-80 °C)
- Freezer (below -20 °C)
- End-over-end rotator
- Vortex mixer
- Gloves
Procedure
Category
Biochemistry > RNA > RNA-protein interaction
Biochemistry > Protein > Immunodetection
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