PMC:13916 / 17071-18479 JSONTXT

{"project":"Colil","denotations":[{"id":"T32","span":{"begin":757,"end":759},"obj":"2197089"}],"namespaces":[{"prefix":"_base","uri":"http://pubannotation.org/docs/sourcedb/PubMed/sourceid/"}],"text":"BRCA1 ribonuclease protection assay and protein analysis\nA BRCA1 ribonuclease protection probe was made using an In Vitro Transcription Kit (Ambion, Inc, Austin, TX, USA). The DNA template spanned part of exon 22, all of exon 23, and part of exon 24 of the BRCA1 gene. Template DNA was incubated for 45min at 37°C with (α-32P)-uridine triphosphate and T7 polymerase in the presence of buffer and nucleotides. DNA template was removed by ribonuclease-free deoxyribonuclease incubation at 37°C for 30min. The reaction was stopped by the addition of 0.5 mol/l ethylenediaminetetra-acetic acid, and the labeled probe was purified on a 5% polyacrylamide gel. Sample RNA (20μg total RNA) was coprecipitated with the BRCA1 probe and the cyclophilin control probe [32], resuspended in Hyb-speed RPA (Ambion) hybridization buffer at 95°C, and then incubated at 68°C for 10min. Ribonuclease was added and the sample was incubated for 45min at 37°C. Protected fragments were precipitated, and resuspended in loading buffer, followed by separation on a 5% polyacrylamide-urea gel, and exposed to X-ray film.\nBRCA1 protein was analyzed by Western blot analysis. Whole cell lysate (50 μg) was loaded onto a 6% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel, transferred to nitrocellulose, and hybridized with an anti-BRCA1 (Ab-1; Oncogene Research, Cambridge, MA, USA) antibody as previously described [10]."}