加入收藏
全国免费热线:400-7780-786

RBC药物筛选实验的常见问题

2019-12-27

化合物的准备工作:

1,化合物的量:0.5-3mg

2,装化合物的管子体积足够大,保证可以放下溶解到10 mM的溶液。

3,如果化合物种类比较多,需要适量的加工费。

实验报告时间:

收到化合物的3周左右。

实验须知:

For each assay in a given order, we will test three types of controls to make sure the assay is consistent from run to run. The DMSO only reactions are used as full activity control. The EDTA only reactions are used as the no or zero activity control. In addition, we includes one standard control compound in 10-dose IC50 format for every target in every order, including single dose duplicate measurements. All control results are included in your report.

Data Delivery 

The raw data, % enzyme activity, and control compound IC50 values will be reported in Excel format for single dose duplicate assays. For IC50 orders, raw data, IC50 values and curve fitting will be delivered in Excel format.

Assay conditions, target, and substrate information are available upon request. Requirements for this information should be noted prior to the commencement of the study.

实验所需试剂和反应步骤:

试剂: 

碱反应缓冲液;20 mM Hepes(pH 7.5),10 mM MgCl2、1 mM EGTA,0.02%Brij35、0.02 mg / ml BSA,0.1 mM Na3VO4、2 mM DTT,1%DMSO 


*需要的辅因子分别添加到每个激酶反应中 


反应步骤:


1.在新鲜制备的基础反应缓冲液中制备指示的底物


2.将所需的辅因子递送至上述底物溶液中


3.将指示的激酶递送至底物溶液中并轻轻混合


4.通过声学技术(Echo550)将DMSO中的化合物递送至激酶反应混合物中;纳升范围),在室温下混和20分钟


5.将33P-ATP(比活为10Ci/l)送入反应混合物中以引发反应。


6.将激酶反应在室温下孵育2小时


7.将反应点到P81离子交换纸上。


.通过滤膜结合法检测激酶活性。



加载中
您是第 1773788 位访客!