A nonradioactive method for detecting DNA-binding activity of nuclear transcription factors.
- Author:
Ning, ZHANG
;
Yongjian, XU
;
Zhenxiang, ZHANG
;
Weining, XIONG
- Publication Type:Journal Article
- MeSH:
Chemiluminescent Measurements;
DNA-Binding Proteins/*analysis;
DNA-Binding Proteins/genetics;
Electrophoretic Mobility Shift Assay;
NF-kappa B/*analysis;
NF-kappa B/genetics;
NF-kappa B/metabolism;
Rats, Sprague-Dawley;
*Trans-Activation (Genetics);
Trans-Activators/analysis;
Trans-Activators/genetics;
*Transcription, Genetic
- From:
Journal of Huazhong University of Science and Technology (Medical Sciences)
2003;23(3):227-9
- 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.
