ADAM-12 (Recombinant)

Frequently Asked Questions

What molecular weight does recombinant ADAM-12 run at on SDS-PAGE and Western blot?

The full-length ADAM-12 protein (UniProt O43184) has a predicted MW of ~100 kDa from its 909-amino-acid sequence, but our HEK293-expressed recombinant typically migrates at ~120–130 kDa under reducing SDS-PAGE conditions. The upward shift reflects N-linked glycosylation added during mammalian expression — a pattern not replicated in E. coli systems. Under non-reducing conditions, disulfide-stabilized forms may migrate anomalously. If you are running this alongside RP-ADAM12 antibody blots, expect the glycosylated band at ~120–130 kDa.

Which isoform and processing form of ADAM-12 is this recombinant — full-length, prodomain removed, soluble ectodomain?

This recombinant corresponds to the soluble ectodomain of ADAM-12 (the predominant shed form), spanning the metalloproteinase, disintegrin, cysteine-rich, and EGF-like domains, with the prodomain removed to yield the active enzyme. The transmembrane segment and cytoplasmic tail are absent. This matches the ADAM-12 form most commonly detected in conditioned media and serum-based functional studies. Researchers studying full-length membrane-anchored ADAM-12 topology should note this distinction when designing cell-surface localization or co-immunoprecipitation experiments.

What substrates can I use to measure ADAM-12 metalloproteinase activity in a fluorescence assay?

ADAM-12 cleaves fluorogenic peptide substrates based on the consensus ADAM cleavage motif. A well-validated choice is the quenched fluorescent peptide Mca-KPLGL-Dpa-AR-NH2 (excitation ~320 nm, emission ~405 nm), used widely for ADAM family activity assays. IGFBP-3 and IGFBP-5 are documented protein substrates if you prefer a gel-based or ELISA readout. Run reactions in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl2, at 37°C. Confirm zinc dependence with 10 mM EDTA as a negative control.

What is a good starting concentration of recombinant ADAM-12 for an in vitro activity assay, and how should I optimize it?

A practical starting point is 50–200 nM recombinant ADAM-12 with 5–20 µM fluorogenic substrate in a 100 µL reaction volume, incubated at 37°C for 30–60 minutes. Titrate enzyme from 25 to 400 nM while holding substrate constant to establish the linear range. Avoid BSA carriers in the assay buffer, as they can quench fluorescence signals. If using inhibitor IC50 studies, fix enzyme at the lowest concentration giving a robust signal (typically ~50 nM) to avoid substrate depletion artifacts during the assay window.

Does the storage buffer for recombinant ADAM-12 interfere with common metalloproteinase activity assays?

The supplied storage buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl2 — is generally compatible with fluorogenic peptide assays at typical dilutions (1:10 or greater into assay buffer). The 10% glycerol is carried over only in trace amounts at working dilutions and does not inhibit ADAM-12 activity at these levels. The 5 mM CaCl2 supports metalloproteinase stability. Do not dilute into EDTA- or EGTA-containing buffers; chelation of Ca²⁺ and Zn²⁺ will inactivate the enzyme irreversibly.

Can I use recombinant ADAM-12 as a positive control for Western blot with the RP-ADAM12 rabbit polyclonal antibody?

Yes — this is a primary validated use case. REC-ADAM12 and RP-ADAM12 are produced and cross-validated in the same lab, so the antibody is confirmed to recognize the recombinant protein on Western blot. Load 50–100 ng of REC-ADAM12 per lane alongside your cell lysates; the glycosylated ectodomain band at ~120–130 kDa serves as a clean size marker. This pairing is also useful for antibody lot-to-lot consistency checks. See the RP-ADAM12 product page (/anti-adam-12-rabbit-polyclonal-antibody) for recommended antibody dilutions and blocking conditions.

How much recombinant ADAM-12 should I load as a Western blot positive control, and will it cause signal saturation?

For standard chemiluminescent detection with RP-ADAM12, 50 ng per lane gives a strong, non-saturated band under typical ECL exposure conditions. If you are titrating antibody concentration or comparing signal across low-abundance lysate samples, a 25–50 ng range keeps the positive control within the linear detection window and avoids blooming that could obscure adjacent lanes. For fluorescent Western systems (e.g., LI-COR), 25 ng is usually sufficient. Run a small titration (10, 25, 50, 100 ng) on your first experiment to calibrate your specific detection setup.

How should I handle, aliquot, and store recombinant ADAM-12 to maintain activity over time?

The protein is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl2. Store at −20°C in single-use aliquots immediately upon receipt; the glycerol concentration supports stability at this temperature without requiring −80°C. Repeated freeze-thaw cycles progressively reduce metalloproteinase activity — plan aliquot sizes around your typical single-experiment usage. Once thawed, keep on ice and use within the same working session. Do not store thawed aliquots at 4°C for more than 24 hours; activity loss is measurable within that window for ADAM family enzymes.