Fluorescence resonance energy transfer-based method for detection of DNA binding activities of nuclear factor kappaB.
The DNA binding protein nuclear factor kappaB (NF-kappaB) and the cellular signaling pathways in which it participates are the central coordinators of many biological processes, including innate and adaptive immune responses, oxidative stress response, and aging. NF-kappaB also plays a key role in diseases, for example, cancer A simple, convenient, and high-throughput detection of NF-kappaB activation is therefore important for systematically studying signaling pathways and for screening therapeutic drug targets. We describe a method based on fluorescence resonance energy transfer (FRET) to directly measure the amount of activated NF-kappaB. More specifically, a double-stranded DNA (dsDNA) probe was designed to contain a pair of FRET fluorophores at the same end of the probe and an endonuclease binding site within the NF-kappaB consensus sequence. The activated NF-kappaB was detected by FRET following the restriction enzyme digestion. Using three different analyte materials--(i) purified recombinant NF- kappaB p50, (ii) nuclear extracts, and (iii) whole cell lysates--we demonstrated that this assay is as sensitive as the traditional, widely used electrophoretic mobility shift assay (EMSA), but much less labor-intensive for measuring NF-kappaB DNA binding activities. In addition, this FRET-based assay can be easily adapted for high-throughput screening of NF-kappaB activation.
He
Hua-Jun
HJ
National Institute of Standards and Technology, Gaithersburg, MD 20899, USA.
Pires
Rick
R
Zhu
Tie-Nian
TN
Zhou
Anhong
A
Gaigalas
Adolfas K
AK
Zou
Sige
S
Wang
Lili
L
D002457:Cell Extracts; D004247:DNA / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D004720:Endonucleases / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D016328:NF-kappa B / Q000032:analysis / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism
D002457:Cell Extracts; D016328:NF-kappa B; D011994:Recombinant Proteins; D004247:DNA; D004720:Endonucleases