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ACVR2A GST Tag Protein, Human

ACVR2A GST Tag Protein, Human

Catalog Number: UA085030 Brand: UA BIOSCIENCE
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Product Details

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.
2. Kosaki, R., et al. (1999). Characterization and mutation analysis of human ACVR2B in patients with left-right axis malformation. American Journal of Human Genetics, 65(3), 730-736.
3. Magga, J., et al. (2019). Systemic Blockade of ACVR2B Ligands Protects Myocardium from Acute Ischemia-Reperfusion Injury. Molecular Therapy, 27(4), 836-849.

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)


 

Picture

Bioactivity

The specific activity of ACVR2A GST Tag Protein was determined to be >1.5 pmol/min/ug as per activity assay protocol.

SDS-PAGE

2μg (R: reducing condition, N: non-reducing condition).

RP-HPLC

The purity of ACVR2A GST Tag Protein, Human is more than 90% determined by RP-HPLC.