ADAM-2 (Recombinant)

Frequently Asked Questions

What molecular weight should I expect for recombinant ADAM-2 on SDS-PAGE or Western blot?

The ADAM-2 polypeptide backbone is 735 amino acids, giving a predicted unglycosylated MW of ~83 kDa. Because this recombinant is expressed in HEK293 cells, N-linked glycosylation adds approximately 10–20 kDa of heterogeneous glycan mass, so expect a diffuse band running at roughly 90–105 kDa under denaturing, reducing conditions. The band may appear slightly smeared rather than a sharp line — this is consistent with native glycosylation and is not a purity artefact. Purity is confirmed >95% by SDS-PAGE prior to release.

Is recombinant ADAM-2 a processed mature form or does it include the prodomain?

The recombinant ADAM-2 (REC-ADAM2) is produced as a proprotein that undergoes furin-mediated cleavage in the HEK293 secretory pathway, consistent with native ADAM processing. The released form retains the metalloprotease-like, disintegrin-like, cysteine-rich, and EGF-like ectodomains, but the prodomain is cleaved. The transmembrane anchor is absent, yielding a soluble ectodomain fragment. If your application requires the prodomain — for example, studying prodomain-mediated inhibitory interactions — this format is not appropriate and you should contact us to discuss alternatives.

Does ADAM-2 have metalloprotease enzymatic activity, and what substrate should I use in an activity assay?

ADAM-2 is non-catalytic. Its metalloprotease-like domain lacks the canonical HExxHxxGxxH zinc-chelating motif residues required for proteolysis, so it does not cleave fluorogenic peptide substrates such as Mca-RPPGFSAFK or generic quenched ADAM substrates. Do not use this recombinant in a protease activity assay expecting cleavage readout. Its functional value is structural and adhesion-mediated: it supports integrin-binding studies, fertilin heterodimer reconstitution with ADAM-1, and sperm–egg adhesion modelling. Design binding or pull-down assays, not enzymatic assays, when working with REC-ADAM2.

What buffer conditions are recommended for ADAM-2 binding or pull-down assays?

REC-ADAM2 is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol. For binding or co-immunoprecipitation assays, this buffer is a reasonable baseline. If you need to dilute into a cell-culture-compatible system, PBS pH 7.4 with 0.1% BSA works well to prevent surface adsorption at low protein concentrations. Avoid prolonged incubation below pH 6.5 or above pH 8.5, which can destabilize the disintegrin-like domain. Do not add EDTA — while ADAM-2 is non-catalytic, chelation may perturb the structural metal coordination in the metalloprotease-like fold.

How much recombinant ADAM-2 should I load as a positive control for Western blot?

For a clean positive-control lane alongside the matched antibody RP-ADAM2, load 50–100 ng of REC-ADAM2 per well on a 4–12% Bis-Tris gel. This consistently produces a detectable band at 90–105 kDa with RP-ADAM2 at standard dilutions (1:500–1:2000). Loading more than 200 ng can cause signal saturation and may obscure the glycosylation-related smear. Including this lane in every blot run is particularly useful when working with lysates from sperm or testicular tissue, where ADAM-2 expression is abundant but background can be high.

Can I use REC-ADAM2 to validate the RP-ADAM2 rabbit polyclonal antibody on Western blot?

Yes — this is one of the most straightforward uses of REC-ADAM2. The matched antibody RP-ADAM2 (/anti-adam-2-rabbit-polyclonal-antibody) was raised and validated against the same recombinant protein, so the combination gives a reliable positive-control signal at 90–105 kDa. Load 50–100 ng of REC-ADAM2, run alongside your experimental samples, and use RP-ADAM2 at 1:500–1:2000 (HRP-conjugated secondary, standard ECL detection). This pairing is also appropriate for antibody lot-to-lot consistency checks and for benchmarking new detection systems.

What is the shelf life of recombinant ADAM-2 and how should I handle it to avoid activity loss?

REC-ADAM2 is stable for at least 12 months from the date of receipt when stored at –20°C in the supplied single-use aliquots. Repeated freeze-thaw cycles degrade the disintegrin-like and cysteine-rich domains — keep aliquot sizes matched to your typical experiment volume. Once thawed, hold on ice and use within 8 hours; do not re-freeze. If you need to work at room temperature for more than 30 minutes, supplement with 0.1% BSA to reduce surface-mediated aggregation. Endotoxin is confirmed <0.1 EU/µg by LAL, so the material is suitable for cell-based adhesion assays without additional depyrogenation.

Is recombinant ADAM-2 suitable for reconstituting the Fertilin heterodimer with ADAM-1 in vitro?

REC-ADAM2 is a practical starting point for Fertilin (PH-30) heterodimer reconstitution because it is produced in HEK293 cells, which support the disulfide-bond formation and N-linked glycosylation that are required for stable ADAM-1/ADAM-2 complex assembly. Published reconstitution studies typically mix equimolar amounts of the two ectodomains (0.5–2 µM each) in PBS pH 7.4 with 0.5 mM CaCl₂ at 4°C for 2–4 hours before downstream pull-down or SPR analysis. Verify heterodimer formation by native PAGE or size-exclusion chromatography before use in adhesion or integrin-binding experiments.