Chromatin immunoprecipitation profiling of human HeLa cells after immunoprecipitation of ptb65 associated mRNAs

ABSTRACT: PTB is multifunctional RNA binding protein reported to be involved in splicing, 3' -end processing, stability and translational regulation. Sample preparation methods (Gama-Carvalho et al, 2006, submitted); Suspension HeLa cells were grown in DMEM, 10% FCS, Pen/Strep and split 1:2 the day before harvesting. Post-nuclear cytoplasmic extracts were obtained as described (Herbert and Hecht 1999), using 5x107 cells/ml of lysis buffer. mRNAs were isolated by immunoprecipitation from 200l of precleared HeLa cell extract, using the anti-U2AF65 MC3 mAb (Gama-Carvalho, Krauss et al. 1997) or the Bb7 anti-PTB mAb (ATCC) for 2h at 4ºC. Immune complexes were precipitated with a slurry with 50% of protein A/protein G agarose beads (Amersham), blocked with 100g/l of tRNA and RNAse free BSA (Amb

ORGANISM(S): Homo sapiens

SUBMITTER: Margarida Gama-Carvalho 

PROVIDER: E-GEOD-6021 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress