Anti-Meprin-A Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight band should I expect for Meprin-A on Western blot?

Full-length Meprin-A subunit alpha (MEP1A) has a calculated molecular weight of approximately 85 kDa based on its 746-amino acid sequence. However, the protein is heavily glycosylated and typically migrates between 95-110 kDa on reducing SDS-PAGE gels. You may also observe higher-order bands corresponding to incompletely reduced multimeric complexes if reducing conditions are insufficient. The antibody is validated at 1:1000 dilution for Western blot. If you see multiple bands, verify sample reduction with fresh DTT or beta-mercaptoethanol, and consider treating lysates with PNGase F to confirm glycosylation-dependent shifts.

Does Meprin-A exist as multiple isoforms or splice variants I should know about?

The MEP1A gene encodes a single major isoform comprising 746 amino acids. Meprin-A functions as part of heteromeric or homomeric complexes with Meprin-beta subunits in vivo, which can complicate interpretation in co-immunoprecipitation experiments. Unlike some metalloproteinases, no catalytically distinct splice variants of the alpha subunit have been widely reported. However, proteolytic processing can generate membrane-shed or truncated forms, particularly in inflammatory contexts. If you are working with conditioned media or detecting lower molecular weight bands, consider that secreted or cleaved fragments may lack the transmembrane domain and may not be recognized depending on epitope location.

Which tissues or cell lines are best for positive controls when testing this Meprin-A antibody?

Meprin-A is most abundantly expressed in kidney proximal tubule epithelium and intestinal epithelial cells. For Western blot positive controls, whole kidney lysate from human, mouse, or rat is the most reliable choice. Human colon or small intestine tissue lysates also work well. Among cell lines, Caco-2 (human colorectal adenocarcinoma) and HK-2 (human proximal tubule) cells express detectable Meprin-A and are commonly used. Expression is typically brush-border localized, so membrane-enriched fractions may yield stronger signals than whole-cell lysates. For negative controls, consider HeLa or HEK293 cells, which show minimal endogenous MEP1A expression under standard culture conditions.

What dilution should I start with for immunohistochemistry or immunofluorescence with this antibody?

This rabbit polyclonal is validated for Western blot at 1:1000 and has been used successfully in IHC and immunofluorescence applications across the Triple Point Biologics platform. For IHC on formalin-fixed paraffin-embedded tissue, start with a 1:200 dilution and titrate up to 1:500 if background is acceptable. Antigen retrieval is typically required; citrate buffer (pH 6.0) heat-induced epitope retrieval works well for kidney and intestinal sections. For immunofluorescence on fixed cells or cryosections, begin at 1:100 to 1:250. Meprin-A localizes to apical brush-border membranes, so expect punctate or linear apical staining in polarized epithelial cells rather than diffuse cytoplasmic signal.

Will this antibody cross-react with mouse and rat Meprin-A, or is it human-specific?

The antibody is validated for human Meprin-A and shows predicted cross-reactivity with mouse, rat, dog, non-human primate, and pig orthologs based on sequence homology in the immunogen region. MEP1A protein sequences share approximately 75-85 percent identity across these mammalian species, with the catalytic and C-terminal domains being highly conserved. However, cross-reactivity is predicted rather than experimentally confirmed for non-human species by Triple Point Biologics. We recommend titrating the antibody with lysates or tissue from your species of interest and including appropriate positive controls. If you observe weak or no signal in rodent samples, consider that expression levels or glycosylation patterns may differ from human.

Should I use protease inhibitors in my lysis buffer when working with Meprin-A samples?

Yes, Meprin-A is an active zinc metalloproteinase with broad substrate specificity, and it can degrade itself and other proteins in your lysate if not inhibited. Include a metalloprotease inhibitor cocktail containing EDTA or 1,10-phenanthroline in your lysis buffer to chelate zinc and prevent autolysis. Standard serine and cysteine protease inhibitors (PMSF, leupeptin, E-64) are also recommended to preserve overall sample integrity. Work quickly on ice and avoid repeated freeze-thaw cycles, which can activate residual protease activity. If you are specifically studying Meprin-A enzymatic activity, prepare separate aliquots without metalloprotease inhibitors and process them immediately for activity assays.

How should I store this Meprin-A antibody and how long will it remain stable?

Store the antibody at -20°C in single-use aliquots to avoid repeated freeze-thaw cycles, which can reduce titer and increase aggregation. The product ships as 100 micrograms of affinity-purified rabbit polyclonal IgG, typically in PBS with glycerol and a preservative. Under these conditions, the antibody is stable for at least 12 months from the date of receipt. For long-term storage beyond one year or for high-value experiments, consider storing at -80°C. Once thawed, an aliquot can be kept at 4°C for up to one month if sterile technique is maintained. Do not store diluted antibody in working solutions; prepare fresh dilutions in blocking buffer for each experiment.

Can I use this antibody to detect secreted or shed Meprin-A in conditioned media or serum?

Meprin-A can be shed from the cell surface by proteolytic cleavage of its transmembrane domain, releasing a soluble form into extracellular fluids. This antibody, raised against a large portion of the ectodomain, should in principle detect shed Meprin-A in conditioned media, urine, or serum. However, sensitivity may be lower than for membrane-bound forms due to dilution and potential epitope masking. Concentrate conditioned media by ultrafiltration or immunoprecipitation before Western blot analysis. Note that serum and urine contain endogenous proteases and inhibitors that may degrade or mask Meprin-A, so add protease inhibitors immediately upon sample collection and process samples promptly. Positive detection will depend on expression levels in your cell system.