RIPK2 Flag Tag Protein, Human

RICK,RIP2,CARDIAK,CARD3

Datasheet COA

价格￥3,020.00 （供应商现货： 3-5个工作日）

货号 UA085022

规格

10μg50μg100μg

数量

产品介绍

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产品规格

物种

Human
分子别名

RICK, RIP2, CARDIAK,CARD3
Accession

O43353
表达序列

Met1-Ala305 with Flag Tag at the N-Terminus
表达宿主

Baculovirus-InsectCells
分子量

35-40kDa (Reducing)
纯度

＞90% by SDS-PAGE,＞95% by HPLC
活性

The specific activity of RIPK2 Flag Tag Protein, Human was determined to be >35 pmol/min/ug.
标记

Unconjugated
标签

Flag 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. Kobayashi, K., et al. (2002). Nod2-dependent regulation of innate and adaptive immunity in the intestinal tract. Science, 307(5710), 731-734.
2. Watanabe, T., et al. (2014). Nucleotide-binding oligomerization domain 1 acts in concert with the cholecystokinin receptor agonist, cerulein, to induce IL-33-dependent chronic pancreatitis. Mucosal Immunology, 7(1), 123-134.

背景介绍

RIPK2 (Receptor-Interacting Protein Kinase 2), also known as RICK or CARDIAK, is a serine/threonine kinase that plays a pivotal role in innate and adaptive immune signaling. It functions as a crucial adaptor and signaling kinase downstream of two distinct classes of pattern recognition receptors (PRRs): NOD1 and NOD2 (Nucleotide-binding oligomerization domain-containing proteins). Structurally, RIPK2 contains an N-terminal kinase domain and a C-terminal caspase activation and recruitment domain (CARD), which mediates its homotypic interactions with the CARD domains of NOD1/NOD2.
Given its central role in NOD signaling, dysregulated RIPK2 activity is strongly implicated in the pathogenesis of chronic inflammatory diseases. Genome-wide association studies have linked RIPK2 variants to inflammatory bowel disease (IBD; Crohn's disease and ulcerative colitis), multiple sclerosis, and Blau syndrome. Consequently, RIPK2 has emerged as a promising therapeutic target for these conditions, with significant efforts focused on developing selective small-molecule inhibitors to modulate its kinase activity and disrupt pathogenic signaling.

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RIPK2活性测定，该试剂盒用于定量检测RIPK2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.RIPK2 Flag Tag Protein, Human
4.ADP-Glo Kinase Assay (Promega, Catalog # V6930)
5.底物: Myelin basic protein (MBP) (Sinobiological, Catalog #M42-51N)
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 0.5 mg/mL
80

2.用激酶检测缓冲液（1x）将RIPK2稀释至67 μg/mL, 33.5 μg/mL and 16.75 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 RIPK2 Flag Tag Protein, Human was determined to be >35 pmol/min/ug.
电泳
- 2μg (R: reducing condition, N: non-reducing condition).
体积排阻色谱(SEC-HPLC)
- The purity of RIPK2 Flag Tag Protein, Human is more than 95% determined by SEC-HPLC.