Anti-MASP-2 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight band should I expect for MASP-2 on Western blot?

Full-length MASP-2 runs at approximately 76 kDa under reducing conditions, corresponding to the uncleaved zymogen form most abundant in serum and cell lysates. Upon activation, MASP-2 is cleaved into a heavy chain (approximately 55 kDa) and a light chain (approximately 27 kDa) that remain disulfide-linked. Under non-reducing conditions, you may observe the 76 kDa form plus higher-molecular-weight complexes if MASP-2 is associated with MBL or ficolins. The antibody recognizes the zymogen; confirm epitope location if you need to detect specific cleaved fragments.

What is the recommended starting dilution for MASP-2 Western blot?

Start at 1:1000 dilution in blocking buffer for Western blot of human serum or liver lysate, which express MASP-2 at physiologically relevant levels. MASP-2 circulates at approximately 400-500 ng/mL in normal human serum, so load at least 5-10 µg total serum protein per lane for reliable detection. For recombinant MASP-2 or overexpression lysates, you may titrate up to 1:2000. Hepatocyte or HepG2 lysates typically require the 1:1000 starting point. Optimize exposure time before adjusting dilution, as MASP-2 is not a high-abundance target in most samples.

Does this antibody cross-react with mouse or rat MASP-2?

Human MASP-2 shares approximately 80% sequence identity with mouse and 78% with rat orthologs, primarily in the serine protease and CUB domains. Cross-reactivity is predicted but not yet validated in our hands. If working with rodent samples, run a positive control (recombinant mouse MASP-2 or mouse serum known to contain MASP-2) alongside your experimental samples. Expect the band near 75-76 kDa. Some polyclonal antibodies raised against human MASP-2 do recognize rodent forms; empirical testing is required to confirm performance in your system.

What sample types and lysis buffers work best for detecting endogenous MASP-2?

MASP-2 is a secreted protein, so serum, plasma, and conditioned media from hepatocytes are the richest sources. For tissue lysates, liver expresses the highest levels. Use RIPA or NP-40-based lysis buffers with protease inhibitors; avoid high concentrations of reducing agents during lysis if you wish to preserve MASP-2 complexes with MBL or ficolins. EDTA in sample buffer will dissociate calcium-dependent lectin interactions. For cell lysates, HepG2 or primary hepatocytes are suitable models. MASP-2 is not abundant in most non-hepatic cell lines, so consider spiking recombinant protein as a positive control.

Can this antibody distinguish between MASP-2 isoforms or splice variants?

Human MASP-2 has two main isoforms: the full-length form (686 amino acids, NM_006610) and a shorter splice variant called MAP19 or sMAP (formerly called small MBL-associated protein), which lacks the serine protease domain and functions as a competitive inhibitor. Without epitope mapping data, assume this polyclonal antibody was raised against a region present in full-length MASP-2. If MAP19 shares that epitope, you may detect a band near 19 kDa. Run recombinant MAP19 as a control if distinguishing isoforms is critical to your experiment.

What controls should I include when using this MASP-2 antibody for the first time?

Include normal human serum (5-10 µg protein per lane) as a positive control; MASP-2 circulates constitutively. Recombinant human MASP-2 (if available) provides a defined-MW reference. For a negative control, use serum or lysate from a cell type that does not express MASP-2, or pre-incubate the antibody with blocking peptide if supplied. Consider a no-primary-antibody lane to assess non-specific secondary binding. If studying lectin-pathway activation, include a sample treated with a known MASP-2 inhibitor to confirm specificity of any cleavage or activity-dependent signal.

How should I store the MASP-2 antibody and what is the expected shelf life?

Store the antibody at -20°C in single-use aliquots to avoid freeze-thaw cycles, which can reduce titre and increase aggregation. Polyclonal antibodies in glycerol-containing storage buffer typically remain stable for 12-24 months at -20°C. For short-term use (up to one month), an aliquot may be held at 4°C with sodium azide preservative. Do not store diluted antibody; prepare working dilutions fresh for each experiment. If you observe elevated background or loss of signal over time, thaw a fresh aliquot and compare side-by-side before troubleshooting other variables.

Will this antibody detect MASP-2 in immunohistochemistry or immunofluorescence?

This antibody is validated for Western blot; IHC and immunofluorescence validation is in progress across the TPB catalogue, but confirm protocol compatibility for MASP-2 specifically. For IHC on formalin-fixed paraffin-embedded liver sections, antigen retrieval (citrate buffer, pH 6.0, heat-induced) is typically required. MASP-2 localizes primarily to hepatocyte cytoplasm and is secreted, so extracellular staining may be faint unless tissue was perfused to retain serum. Start at 1:100 to 1:200 dilution for IHC. For IF on cultured hepatocytes, fix with 4% paraformaldehyde and permeabilize with 0.1% Triton X-100.