Anti-MASP-3 Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight band should I expect for MASP-3 on a Western blot?

MASP-3 migrates at approximately 76–78 kDa on reducing SDS-PAGE, corresponding to the full-length serine protease domain-containing form. Keep in mind that MASP-3 is produced by alternative splicing of the MASP1 gene, which also encodes MASP-1 (approximately 79 kDa) and the smaller MAp44 fragment (approximately 44 kDa). These isoforms share N-terminal domains, so depending on antibody epitope location, you may detect cross-reactivity with MASP-1. Our rabbit polyclonal is raised against residues that should discriminate MASP-3, but confirm specificity with recombinant protein or siRNA knockdown controls where critical.

How do I distinguish MASP-3 from MASP-1 since they come from the same gene?

MASP-3 and MASP-1 are splice variants that share identical N-terminal CUB1-EGF-CUB2 domains but diverge in their serine protease domains at the C-terminus. Antibodies targeting the shared N-terminal region will detect both proteins, while C-terminal epitope antibodies provide isoform specificity. On Western blot, MASP-3 runs slightly lower than MASP-1 (76–78 kDa versus approximately 79 kDa), though separation can be subtle. For definitive identification, use lysates with known MASP-3 expression, include recombinant standards, or deplete one isoform and confirm the remaining band corresponds to your target molecular weight.

What is a good starting dilution for this MASP-3 antibody in Western blot?

Start at 1:1000 dilution in blocking buffer (5 percent non-fat milk or BSA in TBST) for Western blot, which is our validated working dilution. MASP-3 is a secreted protease present at low levels in serum and tissue lysates, so you may need to optimize between 1:500 and 1:2000 depending on sample abundance. Use longer exposures or chemiluminescent substrates with extended dynamic range if signal is weak. For plasma or serum samples, load 2–5 microliters per lane; for tissue or cell lysates, 20–50 micrograms total protein is a reasonable starting point.

Does this antibody work in mouse or rat samples for complement research?

Cross-reactivity with mouse and rat MASP-3 is predicted based on sequence homology but not yet validated in our hands. Human MASP-3 shares approximately 75–80 percent identity with rodent orthologs, with conservation highest in the catalytic serine protease domain. If working with mouse or rat samples, test the antibody at 1:500 to 1:1000 and include a positive control (e.g., human recombinant MASP-3 or human serum) on the same blot. Expect the band at a similar molecular weight (approximately 76 kDa). Report your findings; we incorporate user validation data into future product documentation.

What sample types and preparation work best for detecting MASP-3?

MASP-3 is a secreted liver-derived protease abundant in plasma and serum, making these ideal sample types. Use citrate or EDTA plasma to prevent complement activation artifacts; avoid heparin, which can interfere with downstream detection. For cell or tissue lysates, hepatocytes and liver tissue express MASP-3 at detectable levels. Lyse in RIPA or similar buffer with protease inhibitors (serine protease inhibitors like PMSF or aprotinin are critical). MASP-3 circulates as a zymogen and can be cleaved by endogenous proteases, so work quickly on ice and snap-freeze aliquots to preserve the full-length form.

Can this antibody detect activated versus zymogen forms of MASP-3?

This polyclonal antibody is raised against a region spanning residues 46–460, which includes domains present in both zymogen and activated forms of MASP-3. Upon activation, MASP-3 is cleaved into heavy and light chains that remain disulfide-linked; under reducing conditions you may see separate heavy- and light-chain bands instead of the intact 76-kDa form. Non-reducing SDS-PAGE preserves the disulfide-linked dimer and is preferred for distinguishing zymogen from activated protease. To detect activation in functional assays, complement-activating stimuli (e.g., zymosan or mannan) can trigger cleavage in serum samples.

What controls should I include when validating MASP-3 detection?

Include a positive control such as human serum or plasma (diluted 1:50 to 1:200 in sample buffer), which naturally contains MASP-3 at approximately 5–10 micrograms per milliliter. Recombinant human MASP-3, if available, provides a defined molecular-weight standard. For negative controls, use lysate from a cell line with negligible MASP1 gene expression or serum pre-depleted of MASP-3 by immunoprecipitation. Blocking-peptide competition (if the immunogen peptide is available) confirms epitope specificity. Comparing liver versus non-hepatic tissue lysates also helps, since liver is the primary site of MASP-3 synthesis.

How should I store this antibody and how long does it remain stable?

Store the antibody at –20 degrees Celsius as supplied. For frequent use, aliquot into single-use volumes (e.g., 10–20 microliter aliquots) to avoid repeated freeze-thaw cycles, which can reduce titer and increase aggregation. Once thawed, an aliquot can be held at 4 degrees Celsius for up to one month if sodium azide (0.02–0.05 percent final) is added as preservative. Do not store diluted antibody; prepare working dilutions fresh in blocking buffer on the day of use. Under these conditions, polyclonal antibodies typically retain activity for two years from manufacture date. Check the vial label for the specific lot expiration date.