An optimized assay for transcription factor NF-kappaB with dsDNA-coupled microplate.
Colloids Surf B Biointerfaces
; 55(1): 31-7, 2007 Mar 15.
Article
en En
| MEDLINE
| ID: mdl-17194575
ABSTRACT
To develop an EMSA-free assay approach for analyzing the sequence-specific DNA-binding proteins (DBPs), an easy cost-effective dsDNA-coupled plate (dcPlate) was developed in our lab for this purpose. In this paper, the assay conditions of such dcPlate were fully optimized for detecting an important transcription factor, NF-kappaB. The optimized parameters of dcPlate for assay of NF-kappaB were as follows immobilized DNA probe at the concentration of 25 pmol/100 microL-well, incubation time of 90 min for NF-kappaB binding to dcPlate, primary and secondary antibody concentration of 0.1 microL/100 microL dilution, incubation time of 90 min for primary antibody binding to NF-kappaB, temperature of 25 degrees C for the above process, colorimetric developing time for 30 min. After optimization, the signal was improved three times higher than that from not optimized conditions. The linear colorimetric detection ranges of the purified recombinant NF-kappaB p50 and the cell nuclear extract were from 0.59 to 75 ng/well and 0.313 to 10 microg/well, respectively.
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Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
ADN
/
Análisis de Secuencia por Matrices de Oligonucleótidos
/
Subunidad p50 de NF-kappa B
Límite:
Humans
Idioma:
En
Revista:
Colloids Surf B Biointerfaces
Asunto de la revista:
QUIMICA
Año:
2007
Tipo del documento:
Article
País de afiliación:
China