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Protocol for m3G-cap Immunoprecipitation from Nuclear Extracts

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.