Activin RIB/ALK-4 蛋白, Rhesus Macaque (HEK293, Fc)

Activin RIB, also known as activin receptor type-1B (ACVR1B) or ALK-4, is a type I transmembrane serine/threonine kinase receptor that is part of the TGF-β receptor superfamily. Activin binds to a type II activin receptor (ACVR2or ACVR2B) and then recruits ACVR1B^[1][2][3]. Activin RIB/ALK-4 Protein, Rhesus Macaque (HEK293, Fc) is produced in HEK293 cells with a C-Terminal Fc-tag. It consists of 100 amino acids (R27-E126).

ALK4 也称为激活素 A 受体 1b 型 (ACVR1B)，是一种跨膜丝氨酸/苏氨酸激酶激活素 I 型受体，在哺乳动物心脏中高度表达。ALK4 是脊椎动物发育的重要调节因子，在中胚层诱导、原条形成、原肠胚形成、背前部模式形成和左右轴确定中发挥作用^[1][2]。
不同物种的ALK4（ACVR1B）蛋白的氨基酸序列非常稳定，由此得出的结论是，在进化过程中，ALK4仅发生了轻微的改变，无论在人类还是动物中，其功能类似。
激活素与 II 型激活素受体（Acvr2 或 Acvr2b）结合，然后招募 ACVR1B。ALK4（ACVR1B）与激活素II型受体形成激活素受体复合物，将激活素信号从细胞表面转导至细胞质，从而调节胚胎发生、组织稳态、伤口愈合、细胞外基质产生、免疫抑制和癌变等生理和病理过程。受体异二聚化激活 II 型受体激酶，使 I 型受体磷酸化，从而募集并磷酸化受调节的 Smad2 和 3。磷酸化的受调节 Smad 被释放，并与 Co-Smad、Smad4 形成异聚复合物。然后，受调节的 Smad 和 Co-Smad 复合物易位到细胞核，在那里调节许多基因的表达。在哺乳动物中，Acvr1b 由多种类型的上皮细胞表达，包括毛囊间表皮、毛囊的外根鞘 (ORS) 和内根鞘 (IRS)。激活素信号通过 Acvr1b 以旁分泌方式作用于皮肤上皮细胞^[1][2][3]。
ALK4 ( ACVR1B) 是脊椎动物发育的重要调节因子，在中胚层诱导、原条形成、原肠胚形成、背前模式和左右轴确定中发挥作用。ALK4 还调节生理和病理过程，包括胚胎发生、组织稳态、伤口愈合、细胞外基质产生、免疫抑制和癌发生。ALK4 在胰腺肿瘤发生中充当肿瘤抑制基因^[1][2][3][4]。

Recombinant ALK4 (25-5 ng) and recombinant PTP1B together at 3°C resultes in the apparent cleavage of PTP1B. PTP1B is cleaved after 3 min of Activin treatment, suggesting that Activin causes an interaction between ALK4 and PTP1B that leads to the cleavage of PTP1B by ALK4^[5].

Measured by its binding ability in a functional ELISA. When Recombinant Rhesus Macaque ALK-4/Activin RIB Protein is immobilized at 2µg/mL (100 µL/well) can bind Recombinant Human TDGF1. The ED₅₀ for this effect is 18.31 ng/mL.

• 
  Measured by its binding ability in a functional ELISA. When Recombinant Rhesus Macaque ALK-4/Activin RIB Protein is immobilized at 2 µg/mL (100 µL/well) can bind Recombinant Human TDGF1. The ED₅₀ for this effect is 18.31 ng/mL.

Materials
Biotin
Pre-chilled sterile water
Wash buffer: 1X PBS-T
Blocking buffer: 1% BSA in 1X PBS-T
TMB substrate solution: Mix substrate solution A and B at a 1:1 ratio immediately before use
Stop Solution: H₂SO₄
Activin RIB/ALK-4 Protein, Rhesus Macaque (HEK293, Fc) (HY-P77294)
TDGF1 Protein, Human (HEK293, Fc) (HY-P71112)

Procedure
Biotinylation
1. Dissolution of Biotin Reagent: Remove Biotin from -10 to -30°C and immediately dissolve 1 tube of Biotin in 180 µL of cold purified water. Gently pipette to mix and obtain a 10 mM Biotin solution.
2. Calculation of EZ-Link^TM Sulfo-NHS-LC-LC-Biotin, No-Weigh^TM Format Volume: Calculate the volume to be added.
3. Incubation: Incubate at room temperature in the dark for 45 minutes.
4. Quantification of Biotinylated Protein: Use the BCA assay to quantify the protein concentration. The concentration of biotinylated Human TDGF1 is 998.5 µg/mL.
ELISA Detection
1. Coating: Dilute Recombinant Rhesus Macaque Activin RIB protein to 2 µg/mL in 1X PBS, add 100 µL/well, and incubate overnight at 4°C.
2. Washing: Wash the plate with wash buffer (260 µL/well) , once, and pat dry.
3. Blocking: Add 150 µL per well of blocking buffer (1% BSA in 1X PBS-T) , incubate at 25°C with shaking at 450 rpm for 4 hours.
4. Dilution of Biotinylated Human TDGF1: Prepare dilutions at concentrations of 0, 0.02, 0.2, 1, 2, 10, 20, 100, 200, 1000, 2000, 10000, 20000 ng/mL.
5. Add Biotinylated Human TDGF1: Add 100 µL/well, incubate at 25°C with shaking at 450 rpm for 2 hours.
6. Washing: Wash the plate with Wash Buffer (260 µL/well) , five times, with each wash lasting 1 minute, then pat dry.
7. Add Secondary Antibody: Dilute the secondary antibody at a 1:2000 ratio, add 100 µL /well, incubate at 25°C with shaking at 450 rpm for 1 hour.
8. Washing: Wash the plate with Wash Buffer (260 µL/well) , three times, with each wash lasting 1 minute, then pat dry.
9. Add TMB Substrate Solution: Add 100 µL per well, incubate at room temperature in the dark for 15 minutes.
10. Stop Reaction: Add 100 µL of 2 M H₂SO₄ per well.
11. Read Plate: Measure absorbance at 450 nm.
12. Using GraphPad Prism software, plot a curve with the OD value as the vertical axis and the Biotinylated Human TDGF1 protein concentration as the horizontal axis, and calculate the ED₅₀ value.

Rhesus Macaque

HEK293

C-hFc

I2CYX8/NP_001252559.1 (R27-E126)

696587  [NCBI]

Partial

RGIQALLCACTSCLQANYTCETDGACMVSIFNLDGMEHHVRTCIPKVELVPAGKPFYCLSSEDLRNTHCCYTDYCNRIDLRVPSGHLKEPEHPSMWGPVE

Approximately 43-45 kDa due to the glycosylation.

• 
  Greater than 95% as determined by reducing SDS-PAGE.

无菌粉末.

Lyophilized from a 0.22 μm filtered solution of PBS, pH 7.4.

<1 EU/μg, determined by LAL method.

It is not recommended to reconstitute to a concentration less than 100 μg/mL in ddH₂O. For long term storage it is recommended to add a carrier protein (0.1% BSA, 5% HSA, 10% FBS or 5% Trehalose).

Stored at -20°C for 2 years from date of receipt. After reconstitution, it is stable at 4°C for 1 week or -20°C for longer (with carrier protein). It is recommended to freeze aliquots at -20°C or -80°C for extended storage.

Room temperature in continental US; may vary elsewhere.

• [1]. Dev Dyn, et al. Developmental analysis of activin-like kinase receptor-4 (ALK4) expression in Xenopus laevis. . 2005 Feb;232(2):393-8.  [Content Brief]

  [2]. Qian Wang, et al. Activin Receptor-Like Kinase 4 Haplodeficiency Mitigates Arrhythmogenic Atrial Remodeling and Vulnerability to Atrial Fibrillation in Cardiac Pathological Hypertrophy. J Am Heart Assoc. 2018 Aug 21;7(16):e008842.  [Content Brief]

  [3]. Wanglong Qiu, et al. Conditional activin receptor type 1B (Acvr1b) knockout mice reveal hair loss abnormality. J Invest Dermatol. 2011 May;131(5):1067-76.  [Content Brief]

  [4]. G H Su, et al. ACVR1B (ALK4, activin receptor type 1B) gene mutations in pancreatic carcinoma. Proc Natl Acad Sci U S A. 2001 Mar 13;98(6):3254-7.  [Content Brief]

  [5]. Kamil Matulka, et al. PTP1B is an effector of activin signaling and regulates neural specification of embryonic stem cells. Cell Stem Cell. 2013 Dec 5;13(6):706-19.  [Content Brief]