NEK7 GST Tag Protein, Human

NimA-related protein kinase 7,Never in mitosis A-related kinase 7

Datasheet COA

价格￥3,080.00 （供应商现货： 3-5个工作日）

货号 UA085018

规格

10μg50μg100μg

数量

产品介绍

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产品规格

物种

Human
分子别名

NimA-related protein kinase 7, Never in mitosis A-related kinase 7
Accession

Q8TDX7
表达序列

Met1-Ser302 with GST Tag at the N-Terminus
表达宿主

E.coli
分子量

55-70kDa (Reducing)
纯度

＞90% by SDS-PAGE & > 90% by HPLC
活性

The specific activity of NEK7 was determined to be >6.0 nmol /min/mg as per activity assay protocol.
标记

Unconjugated
标签

GST Tag
性状

Liquid
缓冲体系

50mM Tris, 150mM NaCl, pH7.5, 1mM DTT, 10%Glycerol
储存条件

Stable for 12 months upon stored at -80℃ from the date of receipt. And avoid repeated freeze-thaws cycles.
文献引用

1. Shi, H., et al. (2016). NLRP3 activation and mitosis are mutually exclusive events coordinated by NEK7. Nature, 540(7631), 440-445.
2. He, Y., Zeng, M.Y., Yang, D., Motro, B., & Núñez, G. (2016). NEK7 is an essential mediator of NLRP3 activation downstream of potassium efflux. Nature, 540(7631), 123-127.
3. Kim, S., Lee, K., & Rhee, K. (2007). NEK7 is a centrosomal kinase critical for microtubule nucleation. Biochemical and Biophysical Research Communications, 360(1), 56-62.

背景介绍

NEK7 (NIMA-related kinase 7) is a serine/threonine kinase belonging to the NEK family. It plays a crucial and specific role in regulating cell cycle progression, particularly in mitotic entry and spindle assembly. Structurally distinct from other NEK kinases, NEK7 contains an N-terminal catalytic kinase domain and a C-terminal regulatory domain. Its activity is tightly controlled by cell cycle-dependent phosphorylation and its interaction with NEK9. The NEK9/NEK6/NEK7 cascade is a key pathway for mitotic centrosome separation and spindle formation.

操作步骤

实验方法
实验原理：使用ADP-Glo™激酶检测试剂盒进行NEK7活性测定，该试剂盒用于定量检测NEK7反应产生的ADP量。具体步骤如下：首先加入ADP-Glo™试剂以终止激酶反应并消耗剩余ATP；随后加入激酶检测试剂，将ADP转化为ATP，并通过荧光素酶/荧光素反应体系检测新合成的ATP。
实验材料
1. Kinase assay buffer(5X): 200 mM Tris-HCl, pH 7.4, 100 mM MgCl₂ and 0.5 mg/mL BSA, 250 μM DTT
2. Kinase assay buffer(1X): 40 mM Tris-HCl, pH 7.4, 20 mM MgCl₂, 0.1 mg/mL BSA, 50 μM DTT
3. NEK7 GST Tag Protein, Human
4. ADP-Glo Kinase Assay (Promega, Catalog # V6930)
5. 底物: Casein substrate (Sinobiological, Catalog # C03-54N)
6. Solid white multi-well plate (384-well plate) (Corning, Catalog #3572)
7. 读板器（PerkinElmer）
实验步骤
1. 配制底物/ATP混合液，操作如下（以25 μM为例）：
Sample Name
Amount (μL)
10 mM ATP Solution
1
Kinase Assay Buffer III (5x)
79
Substrate at 1 mg/mL
80
1. 用激酶检测缓冲液（1x）将NEK7稀释至15 µg/mL, 7.5 µg/mL和3.75 µg/mL，并在 384 孔板的每个孔中加入3 µL。
2. 通过向每个孔中加入2 µL微升步骤1中配制的检测系统来启动反应。设置一个仅加入 3 µL激酶检测缓冲液（1x）的检测系统作为空白对照。反应总体积为5 µL。将反应在室温（22–25℃）下孵育40分钟。
3. 向反应完成的体系中加入5 µL ADP-Glo Reagent，短暂混匀后在室温（22–25℃）下孵育 40分钟。
4. 加入10 µL Detection Reagent，将板在室温（22–25℃）下孵育30分钟。
5. 分别使用终点模式读取化学发光信号。
6. 计算比活性。
标准曲线
1. 在激酶检测缓冲液（1×）中将ATP和ADP稀释至25 μM。
2. 按下表所示，混合25 μM ATP和25 μM ADP，配制ATP+ADP混合液，并在384孔板的每个孔中加入5 μL。
Well Number
1
2
3
4
5
6
7
8
9
10
11
12
25μM ADP (μL)
100
80
60
40
20
10
5
4
3
2
1
0
25μM ATP (μL)
0
20
40
60
80
90
95
96
97
98
99
100
1. 向反应完成的孔中加入5 μL ADP-Glo试剂，短暂混匀，在室温（22-25°C）下孵育40分钟。
2. 加入10 μL检测试剂，在室温（22-25°C）下孵育30分钟。
3. 在终点模式下分别读取化学发光信号。
4. 检测光信号并建立转换曲线。
Specific Activity (pmol/min/μg) =
ATP (pmol)-Blank
Incubation time(min) ×amount of enzyme (μg)

生物活性
- NEK7 GST Tag Protein, Human: The specific activity of NEK7 was determined to be >6.0 nmol/min/mg as per activity assay protocol.
电泳
- 2μg (R: reducing condition, N: non-reducing condition).
体积排阻色谱(SEC-HPLC)
- The purity of NEK7 GST Tag Protein, Human is more than 95% determined by SEC-HPLC.