Premium
Nonradioactive ribonuclease protection analysis using digoxygenine labeling and chemiluminescent detection
Author(s) -
Wundrack Iris,
Dooley Steven
Publication year - 1992
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.11501301130
Subject(s) - chemiluminescence , microbiology and biotechnology , chemistry , electroblotting , cell culture , ribonuclease , polyacrylamide gel electrophoresis , transcription (linguistics) , gene , chromatography , biology , biochemistry , rna , enzyme , genetics , linguistics , philosophy
A sensitive nonradioactive ribunouclease protection assay is described which we have used to study c‐myc gene transcription and promoter usage in GLC4, a human small cell lung carcinoma cell line with amplified gene. For in vitro transcription we used digoxygenine (DIG)‐rUTP instead of [α‐ 32 P]CTP or [α‐ 32 P]UTP and after polyacrylamide gel electrophoresis the protected probes were transferred to a nylon membrane from Boehringer Mannheim using electroblotting. Subsequently the membrane was analyzed by chemiluminescent detection. Results were obtained after 1 h of exposure and were comparable with those using radioactivity.