ALK4 GST Tag Protein, Human

ACVRLK4,ACVR1B,Activin receptor type-1B

Datasheet COA

价格￥3,360.00 （供应商现货： 3-5个工作日）

货号 UA085031

规格

50μg10μg100μg

数量

产品介绍

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产品规格

物种

Human
分子别名

ACVRLK4, ACVR1B, Activin receptor type-1B
Accession

P36896-1
表达序列

Asn150-IIe505 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,＞90% by HPLC
活性

The specific activity of ALK4 GST Tag Protein was determined to be >20 pmol/min/ug 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. Activin receptor-like kinases: a novel subclass of cell-surface receptors with predicted serine/threonine kinase activity. Oncogene, 8(10), 2879-2887.
2. Genomic structure and cloned cDNAs predict that four variants of the kinase domain of the type I activin receptor are generated by alternative splicing and poly(A) addition. Biochemical and Biophysical Research Communications, 202(1), 48-54.
3. Adenoviral gene transfer allows Smad-responsive gene promoter analyses and delineation of type I receptor usage of transforming growth factor-beta family ligands in cultured human granulosa-luteal cells. Journal of Clinical Endocrinology & Metabolism, 90(1), 342-350.

背景介绍

ALK4 (Activin Receptor-Like Kinase 4), officially designated ACVR1B (Activin A Receptor Type 1B), is a transmembrane serine/threonine kinase receptor belonging to the TGF-β (Transforming Growth Factor-beta) receptor superfamily. It functions as a type I receptor that is essential for transducing signals from activins and several other TGF-β family ligands, including GDFs (Growth Differentiation Factors). ALK4 does not bind ligand independently. It is activated upon recruitment by a ligand-bound type II receptor (ACVR2A or ACVR2B). The type II receptor phosphorylates ALK4 within its GS domain, activating its kinase. Activated ALK4 then phosphorylates the intracellular signaling effectors SMAD2 and SMAD3. Phosphorylated SMAD2/3 complex with SMAD4 and translocate to the nucleus to regulate target gene transcription.

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ALK4活性测定，该试剂盒用于定量检测ALK4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.ALK4 GST Tag Protein, Human
4.ADP-Glo Kinase Assay (UA, Catalog # UA070101)
5.底物: Bovine Casein Protein (Sinobiological, Catalog # C03-54BN)
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ALK4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 ALK4 GST Tag Protein was determined to be >20 pmol/min/ug as per activity assay protocol.
电泳
- 2μg (R: reducing condition, N: non-reducing condition).
反相高效液相色谱(RP-HPLC)
- The purity of ALK4 GST Tag Protein, Human is more than 90% determined by RP-HPLC.