ACVR1C(ALK7) GST Tag Protein, Human

ALK7 (Activin Receptor-Like Kinase 7) , officially designated ACVR1C (Activin A Receptor Type 1C) , is a type I receptor serine/threonine kinase belonging to the transforming growth factor-β (TGF-β) superfamily. The gene is located on human chromosome 2q24.1, with the gene symbol ACVR1C and aliases including ALK7 and ACVRLK7.

The full-length ALK7 protein consists of 493 amino acids and exhibits all characteristic features of TGF-β type I receptors, including: conserved cysteines and a putative glycosylation site in the extracellular region, a transmembrane region, a GS box, and a C-terminal serine/threonine-rich kinase domain in the intracellular region. Through alternative splicing, the human ALK7 gene encodes four distinct proteins: full-length ALK7, truncated ALK7 (tALK7), and two soluble forms (sALK7a and sALK7b). The ALK7 gene comprises at least 9 exons and 8 introns.

Assay protocol

Principle: The ACVR1C (ALK7) assay is performed using the ADP-Glo^TM Kinase Assay kit which quantifies the amount of ADP produced by the ACVR1C (ALK7) 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

2.Kinase Dilution buffer(1X): 40 mM Tris-HCl, pH 7.4, 20 mM MgCl₂, 0.1 mg/mL BSA, 1mM DTT

3.ACVR1C (ALK7) GST Tag Protein, Human（Catalog # UA085091）

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

5.Substrate: Recombinant Human Smad3 Protein (Full Length, GST Tag) (Sinobiological, Catalog # S12-30G)

6.Solid white multi-well plate (384-well plate) (absin, Catalog # abs7311)

7.Plate Reader (PerkinElmer)

Produce

1.Prepare a substrate/ATP mixture as follows (50 μM example).

2. Dilute the ACVR1C (ALK7) 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.