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6.5K Views
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09:11 min
November 23rd, 2019
DOI :
10.3791/60619-v
Chapters
0:04
Title
0:41
Protein Preparation
2:49
Compound Preparation
3:44
Reagents for the Assay Preparation
5:15
Activity Assay for One 96 Well Plate
7:34
Results: Color Development, Curve of the TmPPase Percent Activity, and Inhibition Curve
8:46
Conclusion
Transcript
我们的协议作为一种工具,寻找抑制膜结合热磷酸盐的去活性的化合物。这种抑制剂可用于治疗几种寄生虫病。该协议是廉价和简单的生产稳定的颜色很长一段时间,不显示任何干扰,在酶重新激活所需的高磷脂浓度的存在。
准备 10 毫升的再活化缓冲液,在 PH 6.5 时包含 20 毫摩尔 M-E-S,按甘油体积计为 3.5%,以 2 毫摩尔 D-T-T 和 0.05%D-D-M 为单位。准备10毫升的反应混合器,含有200毫摩尔三盐酸盐在PH 8。8毫摩尔氯化镁,333毫摩尔氯化钾和67毫摩尔氯
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Summary
在这里,我们提出了膜结合焦磷酶(来自热托加水红素)抑制剂的筛选方法,其依据是96孔板格式的二恶极蓝色反应。
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