Active Mannose Associated Serine Protease 2 (MASP2) (APA859Hu03)

MASP2 (Mannan-binding lectin serine protease 2) is a serum protease that plays an important role in the activation of the complement system via mannose-binding lectin. The preproprotein of MASP2 is proteolytically processed to generate A and B chains that heterodimerize to form the mature protease, which is able to associate with MBL2. Thus, a functional binding ELISA assay was constructed to detect the association of rhMASP2 with MBL2. Briefly, rhMASP-2 were diluted serially in 10mM Tris-HCl, 1M NaCl, 5mM CaCl2, and 0.05%Triton X-100 (pH 7.4). Duplicate samples of 100uL were then transferred to MBL2-coated microtiter wells and incubated for 2h at 37oC. Wells were washed with PBST and incubated for 1h with anti-MASP-2 pAb, then aspirated and washed 3 times. After incubation with HRP labeled secondary antibody, wells were aspirated and washed 3 times. With the addition of substrate solution , wells were incubated for 15-25 minutes at 37oC. Finally, add 50μL stop solution to the wells and read at 450nm immediately. The binding activity of MASP2 with MBL2 was shown in Figure 1 and this effect was in a dose dependent manner.

Figure. Western Blot; Sample: Recombinant MASP2, Human.

The activity of MASP2 was also measured by its ability to cleaves a thioester substrate Z-Lys-SBzl HCl. The reaction was performed in 0.05 M Tris, pH 8.5 (assay buffer), initiated by addition 50 μL of various concentrations of MASP2 (diluted by assay buffer) to 50 μL of substrate and DTNB mixture (equal volumes mixed by 0.4 mM substrate and 0.4 mM DTNB). The final well serves as a negative control with no MASP2, replaced with 50 μL assay buffer. Incubated at 25℃ for 5min, then read at a wavelength of 405 nm. The specific activity of recombinant human MASP2 is >30 pmol/min/μg.

Figure. SDS-PAGE