A nonradioactive method for detecting DNA-binding activity of nuclear transcription factors.
- Author:
Ning ZHANG
1
;
Yongjian XU
;
Zhenxiang ZHANG
;
Weining XIONG
Author Information
1. Department of Respiratory Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030.
- Publication Type:Journal Article
- MeSH:
Animals;
DNA-Binding Proteins;
analysis;
genetics;
Electrophoretic Mobility Shift Assay;
Luminescent Measurements;
NF-kappa B;
analysis;
genetics;
metabolism;
Rats;
Rats, Sprague-Dawley;
Trans-Activators;
analysis;
genetics;
Transcription, Genetic;
Transcriptional Activation
- From:
Journal of Huazhong University of Science and Technology (Medical Sciences)
2003;23(3):227-229
- CountryChina
- Language:English
-
Abstract:
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.
