IRAK4 His Tag Protein, Human

IRAK-4,IMD67,IPD1,NY-REN-64,REN64

Datasheet COA

价格￥2,600.00 （供应商现货： 3-5个工作日）

货号 UA085033

数量

产品介绍

产品介绍评论(0)

产品规格

物种

Human
分子别名

IRAK-4,IMD67,IPD1,NY-REN-64,REN64
Accession

Q9NWZ3
表达序列

Glu154-Ser460 with His Tag at the C-Terminus
表达宿主

Baculovirus-InsectCells
分子量

33-43kDa (Reducing)
纯度

＞90% by SDS-PAGE,> 85% by HPLC.
活性

The IRAK4 assay is performed using the ADP-Glo TM Kinase Assay kit which quantifies the amount of ADP produced by the IRAK4 reaction. The ADP- Glo TM Reagent is added to terminate the kinase reaction and to deplete the remaining ATP, and then the Kinase Detection Reagent is added to convert ADP to ATP and to measure the newly synthesized ATP using luciferase/luciferin reaction. The specific activity is >20 pmol/min/μg.
标记

Unconjugated
标签

His Tag
性状

Lyophilized Powder
缓冲体系

PBS, PH7.4, 5% trehalose
溶解方法

Reconstitute at 0.1-1 mg/ml according to the size in ultrapure water after rapid centrifugation.
储存条件

· 12 months from date of receipt, lyophilized powder stored at -20 to -80℃.
· 3 months, -20 to -80℃ under sterile conditions after reconstitution.
· 1 week, 2 to 8℃ under sterile conditions after reconstitution.
· Please avoid repeated freeze-thaw cycles.
文献引用

1.Wright HJ, Hou J, Xu B, Cortez M, Potma EO, Tromberg BJ, Razorenova OV. CDCP1 drives triple-negative breast cancer metastasis through reduction of lipid-droplet abundance and stimulation of fatty acid oxidation. Proc Natl Acad Sci U S A. 2017 Aug 8;114(32):E6556-E6565.
2.Ackerman L, Acloque G, Bacchelli S, Schwartz H, Feinstein BJ, La Stella P, Alavi A, Gollerkeri A, Davis J, Campbell V, McDonald A, Agarwal S, Karnik R, Shi K, Mishkin A, Culbertson J, Klaus C, Enerson B, Massa V, Kuhn E, Sharma K, Keaney E, Barnes R, Chen D, Zheng X, Rong H, Sabesan V, Ho C, Mainolfi N, Slavin A, Gollob JA. IRAK4 degrader in hidradenitis suppurativa and atopic dermatitis: a phase 1 trial. Nat Med. 2023 Dec;29(12):3127-3136.
3.Kawagoe T, Sato S, Jung A, Yamamoto M, Matsui K, Kato H, Uematsu S, Takeuchi O, Akira S. Essential role of IRAK-4 protein and its kinase activity in Toll-like receptor-mediated immune responses but not in TCR signaling. J Exp Med. 2007 May 14;204(5):1013-24.

背景介绍

IRAK4 (Interleukin-1 Receptor-Associated Kinase 4) is a serine/threonine kinase. Its N-terminal death domain facilitates assembly with MyD88 and IRAK2 into a 6:4:4 helical Myddosome signaling complex, while its C-terminal kinase domain is activated through dimerization and autophosphorylation. A unique tyrosine gatekeeper residue provides a strategic advantage for the development of selective inhibitors. As a central component of the TLR/IL-1R signaling pathway, IRAK4 activates NF-κB and MAPK pathways to regulate innate immune responses. Deficiency in IRAK4 leads to specific susceptibility to pyogenic bacteria (such as Streptococcus pneumoniae) while preserving normal resistance to viral and fungal infections. Clinically, IRAK4 is not only implicated as a causative gene in primary immunodeficiency disorders but also plays a critical role in driving tumor survival in lymphomas harboring MYD88 mutations. Currently, IRAK4-targeted small-molecule inhibitors and PROTAC degraders have advanced into clinical trials for the treatment of rheumatoid arthritis, atopic dermatitis, hidradenitis suppurativa, and B-cell lymphomas. These therapeutic agents achieve precise immunomodulation by either blocking kinase activity or eliminating scaffold functions.

操作步骤

Assay protocol
Principle: The IRAK4 assay is performed using the ADP-GloTM Kinase Assay kit which quantifies the amount of ADP produced by the IRAK4 reaction. The ADP-GloTM Reagent is added to terminate the kinase reaction and to deplete the remaining ATP, and then the Kinase Detection Reagent is added to convert ADP to ATP and to measure the newly synthesized ATP using luciferase/luciferin reaction.
Materials
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.IRAK4 His Tag Protein, Human
4.ADP-Glo Kinase Assay (Promega, Catalog # V6930)
5.Substrate: Myelin Basic Protein (MBP) (Sinobiological, Catalog # M42-51N)
6.Solid white multi-well plate (384-well plate) (Corning, Catalog #3572)
7.Plate Reader (PerkinElmer)
Produce
1.Prepare a substrate/ATP mixture as follows (25 μM example).
Sample Name
Amount (μL)
10 mM ATP Solution
1
Kinase Assay Buffer III (5x)
79
Substrate at 0.5 mg/mL
80

2. Dilute the IRAK4 to 40 µg/mL, 20 µg/mL and 10 µg/mL in Kinase Assay Buffer (1x) and dispense 3 μL into each well of a 384-well plate.
3. Initiate the reaction by adding 2 μL of the detection system prepared in Step 1 to each well. Include a detection system with 3 μL Kinase Assay Buffer (1x) as Blank. The reaction volume is 5 μL.
4. Incubate the reaction at room temperature (22–25℃) for 40 minutes.
5.Add 5 μL of ADP-Glo Reagent to the completed reaction, mix briefly and incubate for 40 minutes at room temperature (22–25℃).
6.Add 10 μL of Detection Reagent and incubate the plate for 30 minutes at room temperature (22–25℃).
7.Read at luminescence, respectively in endpoint mode.
8.Calculate specific activity.
• Standard Curve
Dilute the ATP and ADP to 25 μM in Kinase Assay Buffer (1x).
1.Mix 25 μΜ ATP and 25 μM ADP to form an ATP+ADP solution provided below and dispense 5 μL into each well of a 384-well plate.
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

2.Add 5 μL of ADP-Glo Reagent to the completed reaction, mix briefly and incubate for 40 minutes at room temperature (22–25℃).
3.Add 10 μL of Detection Reagent and incubate the plate for 30 minutes at room temperature (22–25℃).
4.Read at luminescence, respectively in endpoint mode.
5.Detect optical signals and establish conversion curves.
Specific Activity (pmol/min/μg) =
ATP (pmol)-Blank
Incubation time(min) ×amount of enzyme (μg)

实验方法
实验原理：使用ADP-Glo™激酶检测试剂盒进行IRAK4活性测定，该试剂盒用于定量检测IRAK4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.IRAK4 His 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IRAK4稀释至40 µg/mL, 20 µg/mL和10 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)

电泳
- 1μg (R: reducing condition, N:non-reducing condition).
体积排阻色谱(SEC-HPLC)
- The purity of IRAK4 His Tag Protein, Human is more than 85% determined by SEC-HPLC.