A nonradioactive method for detecting DNA-binding activity of nuclear transcription factors.
To determine the feasibility of a nonradioactive electrophoresis mobility shift assay for detecting nuclear transcription factor, double-stranded oligonucleotides encoding the consensus target sequence of NF-kappa B were labelled with DIG by terminal transferase. After nuclear protein stimulated with phorbol 12-myristate 13-acetate (PMA) or PMA and pyrrolidine dithiocarbamate (PDTC) electrophoresed on 8% nondenaturing poliacrylamide gel together with oligeonucleotide probe, they were electro-blotted nylon membrane positively charged. Anti-DIG-AP antibody catalyzed chemiluminescent substrate CSPD to image on X-film. The results showed that nuclear proteins binded specifically to the NF-kappa B consensus sequence in the EMSA by chemiluminescent technique method and the activity of NF-kappa B in PMA group was more than that in PMA + PDTC group. It is suggested that detection of NF-kappa B by EMSA with chemiluminescent technique is feasible and simple, which can be performed in ordinary laboratories.
Zhang
Ning
N
Department of Respiratory Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030.
Xu
Yongjian
Y
Zhang
Zhenxiang
Z
Xiong
Weining
W
D000818:Animals; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D008163:Luminescent Measurements; D016328:NF-kappa B / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D015534:Trans-Activators / Q000032:analysis / Q000235:genetics; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D004268:DNA-Binding Proteins; D016328:NF-kappa B; D015534:Trans-Activators