MASP-2 (Recombinant)

Frequently Asked Questions

What molecular weight does recombinant MASP-2 run at on SDS-PAGE under reducing vs non-reducing conditions?

Under non-reducing SDS-PAGE, active recombinant MASP-2 migrates as a disulfide-linked two-chain heterodimer at approximately 90–95 kDa. Under reducing conditions, the disulfide bond between the A and B chains is broken, yielding two distinct bands: the A chain at ~50 kDa and the serine-protease-containing B chain at ~35 kDa. Purity is >95% by SDS-PAGE. If you observe only a single ~90 kDa band under reducing conditions, the preparation contains a proportion of uncleaved single-chain zymogen — contact us for lot-specific data.

Is this recombinant MASP-2 the zymogen or the activated two-chain form, and how was it activated?

This preparation is the active, two-chain form. The single-chain zymogen precursor (686 aa, UniProt O00187) undergoes cleavage at the Arg448–Ile449 scissile bond to generate the disulfide-linked A chain and catalytic B chain. In vivo this cleavage is mediated by MASP-1; our HEK293-expressed material is processed to the active form during production, confirmed by activity against the chromogenic substrate Boc-Leu-Gly-Arg-pNA. Lot-specific activity data are included with each shipment.

What substrate should I use for a MASP-2 activity assay, and what are recommended buffer conditions?

The well-characterized chromogenic substrate for MASP-2 serine protease activity is Boc-Leu-Gly-Arg-pNA (or the equivalent fluorogenic Z-Lys-SBzl). Run reactions in 50 mM HEPES pH 7.4, 150 mM NaCl, 5 mM CaCl2, 0.05% Tween-20 at 37 °C; calcium is essential for CCP-domain-mediated dimerisation and full catalytic efficiency. The storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol) is compatible if supplemented with CaCl2 to 5 mM prior to assay setup. Monitor absorbance at 405 nm (pNA) or fluorescence at Ex/Em 380/460 nm (fluorogenic).

What starting concentration of recombinant MASP-2 should I use in a complement activity or inhibitor IC50 assay?

For chromogenic substrate assays measuring Km and Vmax, 1–5 nM MASP-2 in a 100 µL reaction volume gives a linear signal window across a 0.1–2 mM substrate range. For inhibitor IC50 determinations, fix MASP-2 at approximately 2 nM and titrate inhibitor across at least 8 concentrations spanning three orders of magnitude. In serum-reconstitution or lectin-pathway reconstitution assays, physiological MASP-2 concentration is ~400–500 ng/mL (~4.5 nM), so dosing at 2–10 nM reflects the relevant in vivo range.

Which natural substrates does active recombinant MASP-2 cleave, and can I use it to generate C4b or C3b in vitro?

Active MASP-2 cleaves complement components C4 (at the C4α Arg756–Ala757 bond) and C2, generating C4b and C2a/C2b — and the resulting C4b2a complex functions as the lectin-pathway C3 convertase, enabling downstream C3b generation. To generate C4b in vitro, incubate recombinant MASP-2 (5–20 nM) with purified human C4 (1–5 µM) in 50 mM HEPES pH 7.4, 150 mM NaCl, 5 mM CaCl2 at 37 °C for 30–60 min. Cleavage can be confirmed by the shift of the C4α chain from ~97 kDa to ~90 kDa under reducing SDS-PAGE.

Can I use recombinant MASP-2 as a positive control for Western blot with the matched RP-MASP2 rabbit polyclonal antibody?

Yes — REC-MASP2 and antibody RP-MASP2 are produced and validated in the same laboratory specifically for this pairing. Under reducing conditions, RP-MASP2 detects the A chain (~50 kDa) and B chain (~35 kDa) as two clean bands; under non-reducing conditions, expect a single band at ~90–95 kDa. This pairing is validated for Western blot and is the recommended positive control configuration for antibody qualification experiments, including those following the guidelines of the International Working Group for Antibody Validation.

How much recombinant MASP-2 should I load per lane as a Western blot positive control for RP-MASP2?

Load 50–200 ng per lane for a clean, unsaturated signal with RP-MASP2 at a 1:1,000–1:2,000 primary antibody dilution on PVDF or nitrocellulose, using standard HRP-conjugated anti-rabbit secondary at 1:5,000–1:10,000. Start at 100 ng if you are titrating alongside serum or cell lysate samples. Because REC-MASP2 is >95% pure by SDS-PAGE, band intensity is highly reproducible between lots, making it suitable for semi-quantitative comparisons or standard curves.

How should I store and handle recombinant MASP-2 after arrival to maintain activity, and what is the shelf life?

Upon receipt, briefly centrifuge the vial and transfer to single-use aliquots if not already done — repeated freeze-thaw cycles reduce serine protease activity measurably. Store at -20 °C in the supplied buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol). Aliquots are stable for at least 12 months at -20 °C. For same-day use, thaw on ice and dilute into assay buffer immediately; do not leave at room temperature for extended periods, as autoproteolytic activity can occur at high concentrations above ~100 nM. Carrier protein (0.1% BSA) may be added for dilutions below 1 nM.