Anti-Meprin-B Rabbit Polyclonal Antibody

Frequently Asked Questions

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

Meprin-B migrates at approximately 95-105 kDa on reducing SDS-PAGE, reflecting the full-length 701-amino acid membrane-bound form with post-translational modifications including N-glycosylation. You may observe additional bands at lower molecular weights corresponding to proteolytic fragments or shed ectodomains, particularly in activated samples. The theoretical mass is around 80 kDa, but glycosylation accounts for the upward shift. If you see multiple bands, consider treating lysates with PNGase F to confirm glycosylation status. Expression levels vary widely by tissue; kidney and intestinal epithelium show highest endogenous expression.

Does this Meprin-B antibody cross-react with Meprin-A subunits?

This antibody is raised against Meprin-B (MEP1B) and does not cross-react with Meprin-A α-subunit (MEP1A), which shares approximately 45% sequence identity. The epitope lies within the Meprin-B-specific regions, ensuring selective detection. However, because Meprin-B can heterodimerize with Meprin-A α-subunits to form functional complexes in vivo, co-immunoprecipitation experiments may pull down both subunits. For Western blot analysis of isolated proteins, you will detect only the β-subunit. If distinguishing membrane-bound Meprin-B from secreted hetero-oligomers matters for your model, consider parallel detection of MEP1A.

What is the recommended starting dilution for Meprin-B Western blot?

Start at 1:1000 dilution in blocking buffer containing 5% non-fat milk or BSA in TBST for overnight incubation at 4°C. Meprin-B expression is relatively low in most cultured cell lines; if signal is weak, you can increase antibody concentration to 1:500 or load more total protein (30-50 µg per lane). Conversely, kidney or intestinal tissue lysates with high endogenous expression may permit dilutions up to 1:2000. Always include a negative control lysate from Meprin-B knockout cells or siRNA-treated samples to confirm band specificity, as non-specific binding near 100 kDa can occur.

Will this antibody work for mouse and rat Meprin-B detection?

Cross-reactivity with mouse and rat Meprin-B is predicted based on high sequence homology in the immunogen region (approximately 85-90% identity), but has not been formally validated by Triple Point Biologics. Many researchers have successfully used this antibody in rodent models, particularly in kidney and colon samples where expression is robust. We recommend validating specificity in your model by testing alongside a Meprin-B knockout or wild-type control. For Western blot, expect similar molecular weight to human (95-105 kDa). If signal-to-noise is poor, empirically optimize dilution starting at 1:500.

How should I prepare cell lysates to preserve Meprin-B enzymatic activity and epitope integrity?

Meprin-B is a membrane-bound metalloprotease; use RIPA or NP-40 lysis buffer supplemented with protease inhibitor cocktail to solubilize without degrading the epitope. Avoid EDTA-containing buffers if you plan parallel activity assays, as Meprin-B requires zinc for catalysis. Include 1 mM PMSF and keep samples on ice during extraction. Ectodomain shedding can occur rapidly in live cells, so lyse immediately after treatment. For immunofluorescence or immunohistochemistry, fix tissue in 4% paraformaldehyde or use frozen sections; antigen retrieval with citrate buffer (pH 6.0) improves epitope accessibility in paraffin-embedded samples.

What positive control tissue or cell line should I use for Meprin-B?

Human kidney cortex lysate serves as an excellent positive control, as Meprin-B is abundantly expressed in proximal tubule epithelial cells. Commercially available kidney lysates or frozen tissue sections work well. Among cell lines, Caco-2 (intestinal epithelium) and HK-2 (proximal tubule) express moderate to high endogenous Meprin-B. HEK293 cells have low baseline expression but can be transfected with MEP1B expression constructs for robust signal. Avoid HeLa or many immune cell lines, which express little to no Meprin-B. Always run a negative control (non-expressing line or knockout) to confirm specificity.

Can this antibody detect Meprin-B after ectodomain shedding or in conditioned media?

The epitope recognized by this antibody spans residues in the ectodomain, so it should detect both the full-length membrane-bound form and shed soluble fragments. If you are analyzing conditioned media or serum, concentrate samples by TCA precipitation or ultrafiltration (10 kDa cutoff) before Western blot, as shed Meprin-B is typically present at low nanomolar concentrations. Expect bands at lower molecular weights (60-80 kDa) corresponding to truncated ectodomains. Note that shedding is context-dependent and influenced by co-expression of sheddases like ADAM10. ELISA-based quantification may be more sensitive than Western blot for soluble Meprin-B.

How should I store this Meprin-B antibody and what is the expected shelf life?

Store the antibody at -20°C in the supplied glycerol-containing buffer; avoid repeated freeze-thaw cycles, which can reduce titer and increase aggregation. For frequent use, aliquot into single-use volumes (10-20 µL) immediately upon receipt. Under these conditions, the antibody remains stable for at least 12 months. Do not store diluted antibody; prepare working dilutions fresh for each experiment. If you observe increased background or loss of signal over time, centrifuge the stock at 10,000 x g for 5 minutes before use to pellet aggregates. Triple Point Biologics has manufactured protease antibodies since 1994 with consistent lot-to-lot performance.