反应 #2200612

反应方程式

cAMP

fluorescein Kemptide

MgCl2

DTT

Kemptide

ATP

→

Fluorescein

溶剂

HEPES

HEPES

反应条件

后处理

1. 1
   其他reaction
2. 2
   其他respectively, with a final reaction volume of 30 ml
3. 3
   其他for a reaction time of 1 hour
4. 4
   workup.ADDITIONwas added to the enzymatic reaction mixture
5. 5
   过滤filter

实验过程

PKA was diluted in 20 mM HEPES, pH 7.4, w/0.05% TX-100 and incubated in a final reaction buffer consisting of 20 mM HEPES, pH 7.4, 0.1 mM cAMP, 1 mM ATP, 5 mM MgCl2, 1 mM DTT with either fluorescein Kemptide (71.25 uM) or OREGON GREEN® Kemptide as the enzymatic substrate at a final concentration of 71.25 or 114.25 mM, respectively, with a final reaction volume of 30 ml, for a reaction time of 1 hour in a 96 well plate format using an opaque white plate. Subsequently, 120 ml of 0.25× Fe(III) working reagent prepared according to Example I was added to the enzymatic reaction mixture and the fluorescence was measured using a 485/538 ex/em filter set. The results are shown in FIGS. 14 and 15.

来源