| High-throughput β-galactosidase and β-glucuronidase Assays Using Fluorogenic Substrates
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Author:
Date:
2013-07-20
[Abstract] β-galactosidase and β-glucuronidase enzymes are commonly used as reporters for gene expression from gene promoter-lacZ or uidA fusions (respectively). The protocol described here is a high-throughput alternative to the commonly used Miller assay (Miller, 1972) that utilises a fluorogenic substrate (Fiksdal et al., 1994) and 96-well plate format. The fluorogenic substrates 4-Methylumbelliferyl β-D-galactoside (for β-galactosidase assays) (Ramsay et al., 2013) or 4-Methylumbelliferyl β-D-glucuronide (for β-glucuronidase assays) (Ramsay et al., 2011) ...
[摘要] β-半乳糖苷酶和β-葡糖醛酸糖苷酶通常用作从基因启动子 - lacZ/em或uidA/em融合（分别）的基因表达的报道分子。本文所述的方案是利用荧光底物（Fiksdal等人，1994）和96孔板形式的通常使用的Miller测定法（Miller，1972）的高通量替代物。荧光底物4-甲基伞形基β-D-半乳糖苷（用于β-半乳糖苷酶测定）（Ramsay等人，2013）或4-甲基伞形基β-D-葡萄糖醛酸苷（用于β-葡糖醛酸糖苷酶测定）（ Ramsay等人，2011）切割以产生荧光产物4-甲基伞形酮。通过冷冻 - 融化和溶菌酶使细胞透化，并且通过荧光微板读数器作为动力学测定连续监测4-甲基伞形酮的产生。然后计算荧光增加的速率，从中推断相对基因表达水平。由于高灵敏度基于荧光的4-甲基伞形酮的检测和所收集的高密度时间点，该测定可以在低水平基因表达的定量中提供增加的准确度。该测定需要小的样品体积和最小的制备时间。本方案中概述的透化条件已经针对革兰氏阴性细菌（特别是大肠杆菌和沙雷氏菌）进行了优化，但是可能适合于具有最小优化的其他生物体。
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| A General Protocol for GST Pull-down
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Author:
Date:
2012-01-20
[Abstract] GST-Pull down assay is an effective way to examine the direct binding of two proteins in vitro. This protocol is based on GST pull down system from GE healthcare, and uses the binding of unplugged/MuSK receptor and Wnt ligand as an example to illustrate the detailed procedure.
[摘要] 胱甘肽-S-芳香基转移酶沉降检测是来研究两个蛋白在体外直接结合一种有效的方法。该方法是基于对胱甘肽-S-芳香基转移酶沉降GE医疗系统，使用结合unplugged/MuSK受体和Wnt信号配体作为一个例子来说明详细过程。
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