A simplified probe preparation for ELISA-based NF-kappaB activity assay.
Nuclear factor-kappaB (NF-kappaB) is critically involved in the transcriptional regulation of many genes and multiple biological and pathobiological processes. To efficiently monitor and to rapidly screen NF-kappaB transcriptional activity, an ELISA-based assay has been increasingly and successfully employed as a new method in a variety of cell lines and experimental models since its first demonstration and recent development. In the ELISA-based assay, NF-kappaB is captured by a double-stranded DNA probe pre-linked on multi-well plates. Typically, the DNA probe contains the double-stranded consensus binding sequence for active NF-kappaB and another double-stranded sequence linking the consensus binding sequence with the plate (linker sequence). Since nuclear factor has no binding activity with single-stranded DNA, we modified the probe construction as containing the double-stranded consensus binding sequence and a single-stranded-linker sequence. Our results show that this kind of probe is highly sensitive and specific for NF-kappaB activity assay, whereas the preparation of this kind of probe is much more convenient. A single-stranded-linker sequence may largely decrease nonspecific protein binding and thus increase the sensitivity of this assay.
Jin
Si
S
Department of Pathophysiology, Tongji Medical College, Huazhong University of Science and Technology; Key Laboratory of Pulmonary disease of Ministry of Health of China, Wuhan 430030, PR China.
Lu
Deqin
D
Ye
Shiqiao
S
Ye
Hong
H
Zhu
Liping
L
Feng
Zuohua
Z
Liu
Shengyuan
S
Wang
Dixun
D
Hu
Qinghua
Q
D001681:Biological Assay / Q000379:methods*; D002460:Cell Line; D042783:Endothelial Cells / Q000378:metabolism*; D004789:Enzyme Activation; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D016328:NF-kappa B / Q000032:analysis / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015345:Oligonucleotide Probes / Q000378:metabolism*
D016328:NF-kappa B; D015345:Oligonucleotide Probes