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.
Accelerating Research
Robert Robinson Avenue,
Oxford Science Park, Oxford
OX4 4GP, United Kingdom
Address
John Eccles HouseRobert Robinson Avenue,
Oxford Science Park, Oxford
OX4 4GP, United Kingdom