NEK7 GST Tag Protein, Human STAR

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 UA085082

数量

产品介绍

产品介绍评论(0)

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

Baculovirus-InsectCells
分子量

55-70kDa (Reducing)
纯度

＞95% by SDS-PAGE & HPLC
活性

1. The specific activity of NEK7 GST Tag Protein, Human (Cat.No.UA085082) was determined to be >7 nmol /min/mg as per activity assay protocol. 2. NEK7 GST Tag Protein, Human (Cat.No.UA085082) at 8nM (4uL/well) can bind CRBN/DDB1 Protein with EC50 of 9.44nM.
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.
A landmark function of NEK7 is its essential role in activating the NLRP3 inflammasome, a critical component of the innate immune system. NEK7 directly interacts with NLRP3 downstream of various danger signals (e.g., ATP, crystalline substances, and pathogens). This interaction is required for NLRP3 inflammasome assembly and the subsequent activation of caspase-1, leading to the maturation and secretion of pro-inflammatory cytokines IL-1β and IL-18 and triggering pyroptosis, a form of inflammatory cell death. This discovery bridges cell cycle regulation with innate immunity. Dysregulation of NEK7 is implicated in auto-inflammatory diseases (e.g., cryopyrin-associated periodic syndromes) and gout, and is a potential target for treating NLRP3-related disorders. In cancer, aberrant NEK7 expression is linked to genomic instability and tumorigenesis due to its role in mitosis.

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

2.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。
3.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。
4.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。
5.加入10 µL Detection Reagent，将板在室温（22–25℃）下孵育30分钟。
6.分别使用终点模式读取化学发光信号。
7.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

3.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。
4.加入10 μL检测试剂，在室温（22-25°C）下孵育30分钟。
5.在终点模式下分别读取化学发光信号。
6.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
- The specific activity of NEK7 GST Tag Protein, Human (Cat.No.UA085082) was determined to be >7 nmol /min/mg as per activity assay protocol.
- Activity assay of NEK7 GST Tag Protein, Human (Cat.No.UA085082) via TR-FRET technology: Incubate NEK7 GST Tag Protein, Human Protein, substrate, corresponding donor and acceptor at 25 °C for 120 min. Finally, read the ratio of fluorescence signals at 620 nm / 665 nm on a TMO Varioskan LUX.
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.