| Hedgehog (Hh) Reporter Activity Assay
|
|
Author:
Date:
2014-07-20
[Abstract] This protocol is for testing responses of a candidate cell line/cell lines to Hh ligands or Hh pathway agonists stimulation. This protocol can also be adapted to screen small molecule libraries or biologics that contain activities to either increase or decrease Hh pathway responses. Canonical Hh signaling activity transcriptionally induces Hh target genes that contain consensus Gli binding element. Hh-responsive cells transiently or stably expressing luciferase protein under the regulation of the Gli promoter element can be used to report stimulus-dependent Hh-pathway activity.
[摘要] 该方案用于测试候选细胞系/细胞系对Hh配体或Hh通路激动剂刺激的反应。 该方案还可以适于筛选含有增加或降低Hh通路反应的活性的小分子文库或生物制剂。 规范Hh信号活动转录诱导含有共有Gli结合元件的Hh靶基因。 在Gli启动子元件的调节下瞬时或稳定表达荧光素酶蛋白的Hh反应性细胞可用于报告刺激依赖性Hh-途径活性。
|
|
|
| Wnt Reporter Activity Assay
|
|
Author:
Date:
2014-07-20
[Abstract] This protocol is for testing responses of a candidate cell line/cell lines to Wnt ligands or Wnt pathway agonists stimulation. This protocol can also be adapted to screen small molecule libraries or biologics that contain activities to either increase or decrease Wnt pathway responses. Canonical Wnt signaling activity transcriptionally induces Wnt target genes that contain concensus TCF/LEF binding element. Wnt pathway activity responsive cells transiently or stably expressing luciferase proteins under the TCF/LEF promoter element can be used to report stimulus-dependent Wnt-pathway activity. We acquired the TopFlash (TCL/LEF-Firefly luciferase) construct from Addgene.
[摘要] 该方案用于测试候选细胞系/细胞系对Wnt配体或Wnt通路激动剂刺激的反应。 该方案还可以适于筛选含有增加或降低Wnt途径反应的活性的小分子文库或生物制品。 规范Wnt信号活动转录诱导含有概括TCF/LEF结合元件的Wnt靶基因。 在TCF/LEF启动子元件下瞬时或稳定表达荧光素酶蛋白的Wnt途径活性应答性细胞可用于报告刺激依赖性Wnt通路活性。 我们从Addgene获得TopFlash（TCL/LEF-Firefly萤光素酶）构建体。
|
|
|
| Helicase Assays
|
|
Author:
Date:
2014-03-20
[Abstract] Helicases are a class of enzymes which are motor proteins using energy derived from ATP hydrolysis to move directionally along a nucliec acid phosphodiester backbone (such as DNA, RNA and DNA-RNA hybrids) and separate two annealed nucleic acid strands. Many cellular processes, such as transcription, DNA replication, recombination and DNA repair involve helicase activity. Here, we provide a protocol to analyze helicase activities in vitro. In this protocol, the DNA helicase protein Merkel cell polyomavirus large T-antigen was expressed in the mammalian cell line HEK293 and immoblized on an IgG resin. The helicase assay is performing while the protein is immoblized on IgG resin.
[摘要] 解旋酶是一类酶，其是使用来自ATP水解的能量沿着核酸磷酸二酯主链（例如DNA，RNA和DNA-RNA杂交体）定向移动并分离两条退火的核酸链的运动蛋白。 许多细胞过程，例如转录，DNA复制，重组和DNA修复涉及解旋酶活性。 在这里，我们提供了一个协议，以分析解旋酶活性在体外。 在该协议中，DNA解旋酶蛋白Merkel细胞多瘤病毒大T抗原在哺乳动物细胞系HEK293中表达并固定在IgG树脂上。 进行解旋酶测定，同时蛋白质固定在IgG树脂上。
|
|
|