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Description
ACVR2A GST Tag Protein, HumanProduct Specification Species Human Synonyms ACVR2, ACTRII, activin A receptor type II Accession P27037 Amino Acid Sequence Arg162 Leu513 with GST Tag at the N Terminus Expression System Baculovirus InsectCells Molecular Weight 55 70kDa (Reducing) Purity 95% by SDS PAGE,90% by HPLC. Conjugation Unconjugated Tag GST Tag Physical Appearance Liquid Storage Buffer 50mM Tris, 150mM NaCl, PH7. 5, 1mM DTT, 10%Glycerol Stability & Storage Stable for 12 months
Product Specification
| Species | Human |
| Synonyms | ACVR2, ACTRII, activin A receptor type II |
| Accession | P27037 |
| Amino Acid Sequence | Arg162-Leu513 with GST Tag at the N-Terminus |
| Expression System | Baculovirus-InsectCells |
| Molecular Weight | 55-70kDa (Reducing) |
| Purity | ＞95% by SDS-PAGE,＞90% by HPLC. |
| Conjugation | Unconjugated |
| Tag | GST Tag |
| Physical Appearance | Liquid |
| Storage Buffer | 50mM Tris, 150mM NaCl, PH7.5, 1mM DTT, 10%Glycerol |
| Stability & Storage | Stable for 12 months upon stored at -80℃ from the date of receipt. And avoid repeated freeze-thaws cycles. |
| Reference | 1. Lee, S.J., et al. (2005). Regulation of muscle growth by multiple ligands signaling through activin type II receptors. Proceedings of the National Academy of Sciences, 102(50), 18117-18122. |
Background
ACVR2B (Activin A Receptor Type 2B) is a transmembrane serine/threonine kinase receptor functioning as a type II receptor for the TGF-β superfamily. It is encoded by the gene located on chromosome 3p22.2 and shares 69% amino acid identity with ACVR2A. Mechanism of Action: ACVR2B serves as a primary activin receptor binding activin A, activin B, inhibin A, myostatin, GDF11, and nodal. Upon ligand binding, it recruits type I receptors (ACVR1B/ALK4, ACVR1/ALK2, or ACVR1C/ALK7) which become phosphorylated and activate downstream SMAD2/3 signaling. Inhibitory SMAD7, recruited via FKBP1A, can prevent SMAD2/3 association with the receptor complex, blocking signal transduction. The receptor also mediates non-SMAD pathways including MAPK and PI3K/AKT signaling.
Protocol
Assay protocol
Principle: The ACVR2A assay is performed using the ADP-GloTM Kinase Assay kit which quantifies the amount of ADP produced by the ACVR2A 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.ACVR2A GST Tag Protein, Human
4.ADP-Glo Kinase Assay (UA, Catalog # UA070101)
5.Substrate: MBP Protein (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 ACVR2A 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
1.Dilute the ATP and ADP to 25 μM in Kinase Assay Buffer (1x).
2.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 |
3.Add 5 μL of ADP-Glo Reagent to the completed reaction, mix briefly and incubate for 40 minutes at room temperature (22–25 ℃).
4.Add 10 μL of Detection Reagent and incubate the plate for 30 minutes at room temperature (22–25 ℃).
5.Read at luminescence, respectively in endpoint mode.
6.Detect optical signals and establish conversion curves.
Specific Activity (pmol/min/μg) = |
ATP (pmol)-Blank |
Incubation time(min) ×amount of enzyme (μg) |
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