ADAM-1 (Recombinant)

Frequently Asked Questions

What molecular weight band should I expect for recombinant ADAM-1 on SDS-PAGE or Western blot?

The full-length ADAM-1 (UniProt Q60813) has a predicted molecular weight of ~90 kDa from its 791-amino-acid sequence, but HEK293-expressed recombinant ADAM-1 typically runs at 95–110 kDa under reducing SDS-PAGE conditions due to N-linked glycosylation. Under non-reducing conditions, disulfide-linked species may appear at higher apparent masses. Purity is confirmed at >95% by SDS-PAGE before release. If you are using the matched antibody RP-ADAM1 for detection, the glycosylated band in this range is the expected positive-control signal.

Is this recombinant ADAM-1 the full-length protein or a processed/cleaved form like the mature ectodomain?

This recombinant represents the mature, processed form of ADAM-1a (Fertilin alpha-a), with the prodomain removed — consistent with the endogenous processing that occurs during sperm maturation. The prodomain acts as an autoinhibitory cysteine-switch; its removal yields the active metalloproteinase and disintegrin domains. The expressed construct retains the disintegrin and metalloproteinase domains critical for integrin binding and zona pellucida interactions. The exact boundaries of the expressed fragment are noted in the datasheet; confirm these match your experimental epitope of interest before proceeding.

What substrates can I use to measure ADAM-1 metalloproteinase activity in a fluorescence assay?

ADAM-1 is a zinc-dependent metalloproteinase; fluorogenic peptide substrates based on the Mca/Dnp quenched-peptide format used broadly for ADAM family members — such as Mca-PLAQAV-Dpa-NH2 or similar sequences — are a reasonable starting point. ADAM-1's in vivo substrates are less well-characterized than ADAM-10 or ADAM-17, so substrate selection should be validated empirically. Run activity assays in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl2 (matching the storage buffer) to avoid buffer-exchange losses and maintain zinc/calcium cofactor requirements.

What is a recommended starting concentration of recombinant ADAM-1 for an in vitro metalloproteinase activity assay?

A starting range of 50–200 nM recombinant ADAM-1 is appropriate for initial fluorogenic substrate assays, with substrate concentrations titrated from 5–100 µM to bracket Km. Because ADAM-1's specific activity toward synthetic peptide substrates has not been as thoroughly benchmarked as ADAM-10 or ADAM-17, include a positive-control metalloproteinase of known activity in the same assay run. Dilute from the stock using assay buffer containing 5 mM CaCl2; omitting calcium can substantially reduce metalloproteinase activity. Avoid BSA-containing diluents unless empirically validated, as BSA can compete in some fluorescence formats.

How should I optimize buffer conditions for recombinant ADAM-1 activity and avoid loss of activity during dilution?

The storage buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl2 — is formulated to stabilize metalloproteinase activity during freeze-thaw. When diluting into assay buffer, maintain CaCl2 at ≥1 mM and keep pH between 7.2 and 7.8; activity drops significantly below pH 6.5. Glycerol can be reduced to 1–2% in the working dilution without major stability impact. EDTA and EGTA are strong inhibitors — even trace contamination from EDTA-containing buffers will chelate zinc and abolish activity. Pre-warm working dilutions to 37°C before adding substrate.

Can I use recombinant ADAM-1 as a positive control for Western blot with the RP-ADAM1 rabbit polyclonal antibody?

Yes — this is a validated pairing. The matched rabbit polyclonal antibody RP-ADAM1 (/anti-adam-1-rabbit-polyclonal-antibody) was raised against ADAM-1 and is guaranteed compatible for Western blot positive-control experiments. Load 50–200 ng of recombinant ADAM-1 per lane alongside your experimental lysates; the antibody should detect the glycosylated band at 95–110 kDa under reducing conditions. This pairing is particularly useful for antibody validation workflows where a defined, high-purity recombinant antigen is needed to confirm specificity before committing to tissue or cell lysate samples.

How much recombinant ADAM-1 should I load for Western blot when validating the RP-ADAM1 antibody?

For a standard Western blot validation lane, load 100 ng of recombinant ADAM-1 as a starting point alongside a titration of 25, 50, and 100 ng to establish the linear detection range with RP-ADAM1. Run under reducing conditions (β-mercaptoethanol or DTT) to resolve the monomer at 95–110 kDa. If signal is saturating at 100 ng, step down to 25–50 ng. Because purity is >95% by SDS-PAGE, the dominant band reflects ADAM-1 and not contaminating proteins, giving a clean positive-control lane for antibody specificity documentation.

What is the shelf life of recombinant ADAM-1 and how should I handle aliquots to preserve activity?

Recombinant ADAM-1 is stable for up to 12 months at -20°C in single-use aliquots in its formulated buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl2). Avoid repeated freeze-thaw cycles — even two cycles can measurably reduce metalloproteinase activity. Upon receipt, spin briefly at 1,000 × g, aliquot into volumes matching your typical experiment, and snap-freeze in liquid nitrogen before returning to -20°C. Once thawed, use within the same working session; do not refreeze. Do not store at 4°C for more than 48 hours, as protease autolysis can occur over time.