RIPK1 GST Tag Protein, Human

RIP,RIP1

Datasheet COA

价格￥3,060.00 （供应商现货： 3-5个工作日）

货号 UA085068

规格

10μg50μg100μg

数量

产品介绍

产品介绍评论(0)

产品规格

物种

Human
分子别名

RIP, RIP1
Accession

Q13546
表达序列

Met1-Ala327 with GST Tag at the N-Terminus
分子量

55-70kDa (Reducing)
纯度

＞90% by SDS-PAGE & HPLC
活性

The specific activity of RIPK1 GST Tag Protein, Human was determined to be > 1.1 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. Clucas, J., & Meier, P. (2023). Roles of RIPK1 as a stress sentinel coordinating cell survival and immunogenic cell death. Nature Reviews Molecular Cell Biology, 24(11), 757-772.
2. Mifflin, L., Ofengeim, D., & Yuan, J. (2020). Receptor-interacting protein kinase 1 (RIPK1) as a therapeutic target. Nature Reviews Drug Discovery, 19(8), 553-571.
3. Ofengeim, D., & Yuan, J. (2013). Regulation of RIP1 kinase signalling at the crossroads of inflammation and cell death. Nature Reviews Molecular Cell Biology, 14(11), 727-736.

背景介绍

RIPK1 (Receptor-Interacting Protein Kinase 1), also known as RIP-1, is a multidomain serine/threonine protein kinase encoded by the human RIPK1 gene (located at 6p25.2). It is a master regulator of inflammatory and cell death signaling pathways, acting as a critical "molecular switch" that determines cell fate (survival or death).
RIPK1 exhibits dual functions—its scaffold function (kinase-independent) and its kinase function—which collectively determine the cell's ultimate fate.
RIPK1 activity is tightly controlled by post-translational modifications including ubiquitination, phosphorylation, and Caspase-8-mediated cleavage. Its dysregulation is implicated in various human diseases.

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RIPK1活性测定，该试剂盒用于定量检测RIPK1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.RIPK1 GST Tag Protein, Human
4.ADP-Glo Kinase Assay (UA, Catalog # UA070101)
5.底物: MBP Protein (Sinobiological, Catalog # M42-51N)
6.MnCl₂(Sigma, CAS No: 7773-01-5)
7.Solid white multi-well plate (384-well plate) (Corning, Catalog #3572)
8.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)
78
Substrate at 0.5 mg/mL
80
MnCl₂
1

2.用激酶检测缓冲液（1x）将RIPK1稀释至30 µg/mL, 15 µg/mL和7.5 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 RIPK1 GST Tag Protein, Human was determined to be > 1.1 nmol /min/mg as per activity assay protocol.
体积排阻色谱(SEC-HPLC)
- The purity of RIPK1 GST Tag Protein, Human is more than 90% determined by SEC-HPLC.