HTRF术语详解 详细 >>
FRET：指荧光共振能量转移，在供体和受体相互靠得很近时，光子能从一个受激发的荧光团（供体）转移到另一个荧光团（受体），并使后者发出荧光。
HTRF：均相时间分辨荧光，Homogeneous Time-Resolved Fluorescence ）是用来检测纯液相体系中待测物的一种常用方法。
背景（Background）：指在没有加入compound时检测到的信号值。
荧光受体（acceptor）：指在荧光团受到激光激发时，能发出特定波长的发射光的基团。HTRF®的能量受体均可为XL665和d2。
资料下载 更多 >>
- New HTRF cellular platform for cell surface receptors' study and screening
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- HTRF Compatible Readers:Unleash your lab's capacity to read HTRF technology
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A homogeneous time-resolved fluorescence-based high-throughput screening system for discovery of inhibitors of IKKbeta-NEMO interaction
By: Gotoh Y, Nagata H, Kase H, Shimonishi M, Ido M.
Genomic Science Laboratories, Dainippon Sumitomo Pharma, Konohana-ku, Osaka 554-0022, Japan.
The nuclear transcription factor NF-kappaB is crucial to the expression of numerous cytokines, enzymes, and cell adhesion molecules, all of which can drive inflammatory and autoimmune disorders such as rheumatoid arthritis. The IKK complex plays the most important role in the signal cascade leading to NF-kappaB activation. Recently, inhibition of the interaction between NEMO (NF-kappaB essential modulator) and the catalytic subunits of IKK, especially IKKbeta, has received particular attention as a possible new therapeutic approach to treatment of inflammatory disorders, and several reports have shown the efficacy of cell permeable NEMO binding domain (NBD)-containing peptides in blocking the IKK/NF-kappaB pathway. In this article, we describe in detail the development and validation of two novel binding assays, a homogeneous time-resolved fluorescence (HTRF)-based assay and an enzyme-linked immunosorbent assay (ELISA)-based assay, suitable for the discovery of small molecules that inhibit IKKbeta-NEMO interaction. Using the HTRF-based assay, we screened approximately 15,000 compounds from our chemical library and eliminated false positive hits by the ELISA-based assay and IKK complex kinase assay. As a result, seven positive hit compounds that inhibit IKK complex activity through inhibition of IKKbeta-NEMO interaction were identified. These hit compounds may have a good potential in the treatment of inflammatory and autoimmune disorders such as rheumatoid arthritis.
编辑: cq
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