ACVR2A His Tag Protein, Human

ACVR2A (Activin A Receptor Type 2A) is a transmembrane serine/threonine kinase receptor that serves as a type II receptor for the TGF-β superfamily. It is encoded by the gene located on chromosome 2q22.3 and is expressed in multiple tissues including brain, testis, intestine, liver, and kidney.

Mechanism of Action: ACVR2A functions as a constitutively active kinase that binds ligands including activin A, activin B, inhibin A, GDF8/myostatin, and GDF11. Upon ligand binding, ACVR2A recruits and phosphorylates type I receptors (ACVR1B/ALK4, ACVR1/ALK2, or ACVR1C/ALK7) within their GS domains. The activated type I receptor then phosphorylates SMAD2 and SMAD3, which complex with SMAD4 and translocate to the nucleus to regulate gene transcription. Inhibitory SMAD7 can block this signaling cascade.

Assay protocol

Principle: The ACVR2A assay is performed using the ADP-Glo^TM Kinase Assay kit which quantifies the amount of ADP produced by the ACVR2A 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.

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 His 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).

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.

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.