PAPP-A2 His Tag Protein, Human

PAPP-A2 (Pregnancy-associated plasma protein-A2) is a secreted metalloproteinase belonging to the pappalysin family, whose structure comprises multiple functional domains that specifically hydrolyze insulin-like growth factor binding proteins 3 and 5 (IGFBP-3/5). By cleaving IGFBPs, PAPP-A2 releases free IGF-1, thereby precisely regulating IGF-1 bioavailability and playing a central role in fetal-placental development, postnatal linear growth, and bone metabolism. Clinical studies have demonstrated that PAPP-A2 gene mutations cause PAPP-A2 deficiency syndrome, characterized by idiopathic short stature, delayed bone age, and a distinctive biochemical phenotype of decreased free IGF-1 with elevated total IGF-1, while recombinant human IGF-1 replacement therapy can significantly improve patient growth; furthermore, abnormal placental PAPP-A2 expression is closely associated with preeclampsia, indicating that it serves not only as a critical regulator of growth and development but also as a potential biomarker for pregnancy complications.

Assay protocol

Principle: As an active protease, recombinant human PAPP-A2 cleaves IGFBP-5 under the conditions described in Activity Assay Protocol.

Materials

1.Assay Buffer: 50 mM Tris, pH 8.0

2.PAPP-A2 His Tag Protein, Human

3.Human IGFBP5 Protein, His Tag (S0A0185)

4.4-20% SDS-PAGE gel

5.5×reducing SDS-PAGE gel buffer

6.SDS-PAGE

Produce

1. Dilute rhPappalysin-2 to 40 µg/mL in Assay Buffer.

2. Dilute rhIGFBP-5 to 200 µg/mL in Assay Buffer (Note: Acetonitrile interferes with assay, dry down rhIGFBP-5 and reconstitute at

200 µg/mL with Assay Buffer if necessary).

3. Combine equal volumes of 40 µg/mL rhPappalysin-2 and 200 µg/mL rhIGFBP-5. Include two blank controls containing Assay Buffer in place of rhPappalysin-2.

4. Incubate reaction vials and one control at 37 °C overnight. Keep the other control at -20 °C during the overnight incubation period.

5. Combine 20 µL from each reaction vial with 5µL 5×reducing SDS-PAGE gel buffer.

6. Analyze the cleavage by SDS PAGE (load 20 µL/lane) followed by protein staining.

实验方法

实验原理：PAPP-A2为活性蛋白酶，可在本活性检测方案规定的反应条件下切割胰岛素样生长因子结合蛋白 5（IGFBP-5）。

实验材料

1.检测缓冲液: 50 mM Tris, pH 8.0

2.PAPP-A2 His Tag Protein, Human

3.Human IGFBP5 Protein, His Tag (S0A0185)

4.4-20% SDS-PAGE gel

5.5×reducing SDS-PAGE gel buffer

6.SDS-PAGE

实验步骤

1.用检测缓冲液将重组人PAPP-A2稀释至40 μg/mL。

2.用检测缓冲液将重组人IGFBP-5稀释至200 μg/mL（注意：乙腈会干扰本实验；若样品中含乙腈，需先将重组人IGFBP-5冻干去除溶剂，再用检测缓冲液复溶并配制成200 μg/mL溶液）。

3.取等体积的40 μg/mL PAPP-A2溶液与200 μg/mL IGFBP-5溶液混合；同时设置两组空白对照，对照组以检测缓冲液替代PAPP-A2蛋白溶液。

4.将反应样品及其中一份对照组样品置于 37 ℃条件下过夜孵育；另一份对照组样品全程置于- 20 ℃冷藏保存。

5.从各反应管中分别取20 μL反应液，与5 μL 5×还原性SDS-PAGE上样缓冲液并混匀。

6.取20 μL混合液上样（每泳道），进行SDS-PAGE电泳；随后通过蛋白染色分析蛋白切割结果。