General considerations
Use RNase free molecular biology grade water for all buffers and RNase free tips and tubes.
Materials and Solutions needed
- PBS: Phosphate buffered saline, pH 7.4
- Protein A or protein G sepharose
- Ice
- IPP buffer: Tris-HCl pH 7.4, 150 mM NaCl, 0.1% NP40
- Phenol/chloroform
- RNA-elution buffer: Tris-HCl pH 7.4, 450 mM NaCl, 0.4% SDS
- 96% ethanol
- 80% ethanol
- RNase inhibitor (e.g. RNasin)
Procedure
- 10 - 20 µg antibody per assay are coupled to protein A or protein G sepharose in PBS at 4°C head over tail (several hours).
- The pellet is washed three times with ice-cold PBS.
- Incubate immobilized antibody with 20 µl nuclear extract in 250 µl IPP buffer for 1 h on a head over tail rotor at 4°C. The buffer provides stringency to avoid non-specific interaction. Generally, non-specific interactions should be controlled with a parallel pull-down assay using protein A/G-sepharose without antibody.
- Wash five times with 1 ml of IPP buffer. After two washes the content of the reaction tube should be transferred to a new one. This step significantly reduces background in pull-down assays.
- The pellet-bound RNA can be isolated by shaking the tube with 250 µl of IPP buffer with one volume of phenol/chloroform and subsequent ethanol precipitation of the aqueous phase. Alternatively, the precipitated RNA-(complex) may be eluted by shaking with 250 µl of RNA-elution buffer. After phenol/chloroform-extraction of the eluate protein and RNA-containing phases are precipitated and subjected to analysis.
- RNA-analysis: native RNA may be analyzed by 3´-terminal pCp-labelling or Northern-Blot.
For more background information refer to Bochnig P et al., 1987.
