Kcat/Km就可以用来确定酶的最适底物，同一个酶对不同底物的Kcat/Km最大可相差100万倍。 当利用蛋白质工程对酶进行改造时，不同突变型的Kcat/Km也是需要测定的参数，用来表征酶催化效率的变化情况。

km的定义是一半最高反应速率时的atp浓度，这样对于不同的激酶反应atp浓度是不一致的。 所以对于ROCK2酶学assay的ATP浓度，先计算Km为25μM，因此应该选择25μM ATP浓度。

2014年7月18日 · We evaluated a large number of JAK inhibitors in enzymatic assays utilizing either 1 mM ATP or Km ATP for the four isoforms as well as in primary cell assays. This data set provided the opportunity to examine individual kinase contributions to the heterodimeric kinase complexes mediating cellular signaling.

2014年6月10日 · Another important improvement over other large screening sets is that we measure the Km for ATP of each kinase and use a concentration of ATP near to the Km for each individual kinase assay, and then measure precise IC 50 values. This gives a much more accurate inhibition value as compared to utilizing the same fixed ATP concentration for all ...

ATP concentration greatly affects the determination of kinase inhibitory activity, given most kinase inhibitors bind to the ATP binding site of the kinase. Cell free (biochemical) assays typically utilize ATP concentrations approximating the ATP Km, however this is frequently far lower than the mM ATP concentrations found in cells.

HotSpot kinase screening service now available at physiologically relevant 1mM ATP for 340 wild type kinases, in addition to existing standard concentrations of 1μM, 10μM or apparent ATP-Km up to 100μM; Specialty panels comprise a selection of kinase-specific mutants or our CDK panel.

2014年5月12日 · We evaluated a large number of JAK inhibitors in enzymatic assays utilizing either 1 mM ATP or Km ATP for the four isoforms as well as in primary cell assays. This data set provided the opportunity to examine individual kinase contributions to the heterodimeric kinase complexes mediating cellular signaling.

该方法利用激酶反应后剩余的ATP，将荧光素转化为氧化荧光素，并发出化学冷光，荧光信号值与激酶活性成反比。 该试剂盒里的重组热稳定荧光素酶 (Ultra-GloTM)，发出稳定的“辉光”型荧光，半衰期大于5个小时，不需要配套进样器的荧光仪，可成批处理大量的微孔板。 Ultra-GloTM发出的稳定荧光可适用于多种检测条件，与特定的缓冲液组成相搭配，可减少来自化合物的自发荧光的干扰。 该检测方法的成本很低，但为了产生大于2倍的信噪比，至少需要50%的转化率，因此不能 …

每个 KES对应的Application Note中，激酶检测使用的ATP浓度是与已发布的其同一类激酶家族ATP Km值范围最接近的浓度。我们建议您使用该ATP浓度来进行激酶滴定和抑制剂滴定实验。

使用1 mm atp可帮助我们了解和预测化合物在生理环境中的特性。 ATP在Km值附近和1 mM条件下，激酶测定中舒尼替尼不同的选择性特征如下图所示。 1 mM ATP下对激酶测定的验证结果见 所有靶激酶页面 。