ALK4 GST Tag Protein, Human

ALK4 (Activin Receptor-Like Kinase 4), officially designated ACVR1B (Activin A Receptor Type 1B), is a transmembrane serine/threonine kinase receptor belonging to the TGF-β (Transforming Growth Factor-beta) receptor superfamily. It functions as a type I receptor that is essential for transducing signals from activins and several other TGF-β family ligands, including GDFs (Growth Differentiation Factors). ALK4 does not bind ligand independently. It is activated upon recruitment by a ligand-bound type II receptor (ACVR2A or ACVR2B). The type II receptor phosphorylates ALK4 within its GS domain, activating its kinase. Activated ALK4 then phosphorylates the intracellular signaling effectors SMAD2 and SMAD3. Phosphorylated SMAD2/3 complex with SMAD4 and translocate to the nucleus to regulate target gene transcription.

Assay protocol

Principle: The ALK4 assay is performed using the ADP-GloTM Kinase Assay kit which quantifies the amount of ADP produced by the ALK4 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.ALK4 GST Tag Protein, Human

4.ADP-Glo Kinase Assay (UA, Catalog # UA070101)

5.Substrate: Bovine Casein Protein (Sinobiological, Catalog # C03-54BN)

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 ALK4 to 30 µg/mL, 15 µg/mL and 7.5 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.