Protocol for m6A Immunoprecipitation from Nuclear Extracts
Materials and solutions needed
RNase-free molecular biology-grade water
PBS: Phosphate buffered saline, pH 7.4
Protein A or protein G sepharose
IPP buffer: Tris-HCl pH 7.4, 150 mM NaCl, 0.1% NP40
RNA-elution buffer: Tris-HCl pH 7.4, 450 mM NaCl, 0.4% SDS
RNase inhibitor (e.g. RNasin)
10 - 15 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 3 times with ice-cold PBS.
Incubate immobilized antibody with 20 µl nuclear extract in 250 µl IPPbuffer 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 5 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.