Quantitative chemiluminescent immunoassay for NF-kappaB-DNA binding activity.
Nuclear factor-kappaB (NF-kappaB) is a ubiquitous redox-sensitive transcription factor involved in the pro-inflammatory response to several factors, including cytokines and oxidative stress. Upon activation, NF-kappaB translocates into the nucleus and binds to specific nucleotide sequences. The cellular responses to inflammatory and stress signals have been implicated in disease conditions, such as atherosclerosis, cancer, diabetes, and Alzheimer's disease. The conventional method for detection of NF-kappaB -DNA binding activity is the electrophoretic mobility shift assay (EMSA), which is time-consuming and non-quantitative. Here, we report (a) development of a rapid, sensitive and quantitative chemiluminescent immunoassay (QCI) for analysis of NF-kappaB DNA-binding activity, and (b) validation of the QCI with the EMSA using nuclear and cytosolic extracts from cultured prostate cancer cells (PC3), rat liver homogenates and human lymphocytes. The QCI for analysis of NF-kappaB DNA binding activity has advantages over the EMSA: (1) Higher speed: 3-5h post sample preparation, (2) Greater sensitivity: 10pg NF-kappaB/well, (3) Quantitative: linear range: 10-1000pg NF-kappaB; r2 = 0.999 (4) High throughput adaptability: 96-well plate format can analyze up to 40 samples in duplicate, (5) SAFETY: No radioactive isotopes, (6) Simplicity, and (7) Capability of measurement of both activated (free) NF-KB which is translocated into the nucleus and total (bound + unbound) NF-kappaB present in the cytosol/cell.
Gupta
Smiti V
SV
Oxford Biomedical Research, 2165 Avon Industrial Drive, Rochester Hills, MI 48309, USA. sgupta@oxfordbiomed.com
McGowen
Richard M
RM
Callewaert
Denis M
DM
Brown
Thomas R
TR
Li
Yiwei
Y
Sarkar
Fazlul H
FH
D000818:Animals; D045744:Cell Line, Tumor; D003600:Cytosol / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D008099:Liver / Q000166:cytology / Q000378:metabolism; D008163:Luminescent Measurements / Q000379:methods*; D008214:Lymphocytes / Q000378:metabolism; D008297:Male; D016328:NF-kappa B / Q000378:metabolism*; D010084:Oxidation-Reduction; D011471:Prostatic Neoplasms / Q000378:metabolism; D051381:Rats; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism
D004268:DNA-Binding Proteins; D016328:NF-kappa B; D014157:Transcription Factors; D004247:DNA