ALK5 GST Tag Protein, Human

AAT5,ACVRLK4,ALK-5,ALK5,SKR4,TGFβR-1,TGFR1

Datasheet COA

价格￥1,180.00 （供应商现货： 3-5个工作日）

货号 UA085034

规格

10μg50μg100μg

数量

产品介绍

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产品规格

物种

Human
分子别名

AAT5, ACVRLK4, ALK-5, ALK5, SKR4, TGFβR-1, TGFR1
Accession

P36897
表达序列

Thr200-Met503 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

The specific activity of ALK5 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. Larsson, J., et al. (2001). Abnormal angiogenesis but intact hematopoietic potential in TGF-beta type I receptor-deficient mice. Development, 128(10), 1863-1873
2. Loeys, B. L., et al. (2005). A syndrome of altered cardiovascular, craniofacial, neurocognitive and skeletal development caused by mutations in TGFBR1 or TGFBR2. Nature Genetics, 37(3), 275-281.
3. Mazerbourg, S., et al. (2004). Identification of receptors and signaling pathways for growth differentiation factor-9 (GDF-9) in ovarian cells. Molecular Endocrinology, 18(9), 2229-2240.

背景介绍

ALK5 (Activin Receptor-Like Kinase 5), officially designated TGFBR1 (Transforming Growth Factor Beta Receptor 1), is the primary type I serine/threonine kinase receptor for TGF-β cytokines. It is encoded by the gene located on chromosome 9q22.33 and is ubiquitously expressed.
Mechanism of Action: TGF-β ligands first bind with high affinity to the type II receptor (TGFBR2). This complex then recruits and phosphorylates ALK5 at its GS domain. Activated ALK5 phosphorylates SMAD2 and SMAD3, which complex with SMAD4 and translocate to the nucleus to regulate transcription of genes involved in cell cycle arrest, extracellular matrix production, epithelial-mesenchymal transition (EMT), and differentiation. Beyond canonical SMAD signaling, ALK5 also activates non-SMAD pathways.
Physiological & Pathological Roles: ALK5 is a master regulator of homeostasis and development. It is essential for embryonic development, as murine knockout is embryonic lethal. In adults, it functions as a tumor suppressor in many epithelial tissues by inhibiting proliferation; loss of its negative regulation contributes to cancer. Conversely, in advanced cancers, ALK5 signaling can promote metastasis via EMT. Germline mutations in TGFBR1 cause Loeys-Dietz syndrome (LDS), a connective tissue disorder characterized by aortic aneurysms. Somatic mutations (e.g., A230V) in the kinase domain are found in endometrial cancer and result in partial loss-of-function and reduced inhibitor sensitivity.

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ALK5活性测定，该试剂盒用于定量检测ALK5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.ALK5 GST Tag Protein, Human
4.ADP-Glo Kinase Assay (Promega, Catalog # V6930)
5.底物: TGFBR1 peptide (Sinobiological, Catalog # T36-58)
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ALK5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 ALK5 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).
体积排阻色谱(SEC-HPLC)
- The purity of ALK5 GST Tag Protein, Human is more than 95% determined by SEC-HPLC.