ADAM-32 (Recombinant)

Frequently Asked Questions

What molecular weight band should I expect for recombinant ADAM-32 on SDS-PAGE or Western blot?

The predicted molecular weight of the 787-amino-acid ADAM-32 polypeptide is approximately 87 kDa, but HEK293-expressed recombinant ADAM-32 typically migrates at 100–115 kDa under denaturing SDS-PAGE conditions due to N-linked glycosylation across multiple extracellular domain sites. Under reducing conditions the band may shift slightly. When using RP-ADAM32 (our matched rabbit polyclonal) for Western blot validation, a single dominant band in this 100–115 kDa range is the expected positive control result. Anomalous migration above 115 kDa may indicate incomplete reduction or intermolecular disulfide artifacts.

Does ADAM-32 have metalloprotease activity and can I use a standard ADAM fluorogenic substrate assay?

No. ADAM-32 is a non-catalytic metalloprotease-like protein — its metalloproteinase domain lacks the conserved zinc-coordinating glutamate (HExxH motif glutamate) essential for proteolytic activity. Standard fluorogenic ADAM substrates such as Mca-KPLGL-Dpa or quenched TNF-alpha peptides will not be cleaved by this recombinant. Functional assays should instead be designed around ADAM-32's disintegrin domain, including integrin-binding assays (e.g., solid-phase integrin capture) or cell-adhesion competition experiments, where the extracellular domain mediates protein–protein interactions rather than substrate cleavage.

What buffer is recombinant ADAM-32 supplied in and is it compatible with integrin-binding assays?

Recombinant ADAM-32 is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, plus 5 mM CaCl2 (standard for ADAM family members). This buffer is generally compatible with solid-phase integrin-binding assays provided you dilute the glycerol to ≤1% in the assay well to prevent non-specific blocking. For SPR or biolayer interferometry, dialyze or buffer-exchange into your running buffer (e.g., HBS-EP+) before use. Avoid Ca2+-chelating agents such as EDTA or EGTA, as divalent cations support ADAM extracellular domain integrity.

What starting concentration of recombinant ADAM-32 should I use in a disintegrin domain integrin-binding experiment?

In solid-phase integrin capture formats, a useful starting range is 0.5–5 µg/mL of recombinant ADAM-32 coated on Ni-NTA or streptavidin plates (depending on your tag/biotinylation strategy). For competition or solution-phase binding assays, titrate from 10 nM to 1 µM to bracket expected Kd values for ADAM disintegrin–integrin interactions, which typically fall in the 10–500 nM range for this subfamily. Run a no-protein background control in the same Tris/NaCl/glycerol buffer to correct for glycerol-associated signal at high protein concentrations.

Can I use recombinant ADAM-32 as a positive control for Western blot with the RP-ADAM32 rabbit polyclonal antibody?

Yes — this is one of the primary validated uses. Recombinant ADAM-32 (REC-ADAM32) and the matched antibody RP-ADAM32 are produced and validated together in the same laboratory. Load 20–50 ng of recombinant ADAM-32 per lane alongside your cell lysate samples; this quantity reliably produces a clear band at 100–115 kDa with RP-ADAM32 at standard WB dilutions (typically 1:500–1:2,000). This pairing confirms antibody performance and provides a molecular-weight reference that accounts for glycan-shifted migration — a common source of confusion when comparing to predicted mass databases.

How much recombinant ADAM-32 should I load for a Western blot positive control lane?

20–50 ng per lane is the validated range when using RP-ADAM32 as the primary antibody. At 20 ng you should see a clean, unambiguous band at 100–115 kDa after ECL detection with standard HRP-conjugated secondaries. Loading above 100 ng risks satellite banding from glycoprotein heterogeneity and can bleed into adjacent lysate lanes. If your detection system uses fluorescent secondary antibodies, start at the lower end (20 ng) and titrate up. Confirm band identity by including a no-primary negative control in parallel.

What is the processing or isoform status of this recombinant — is it the pro-form or mature ectodomain?

This recombinant represents full-length human ADAM-32 (787 residues; UniProt Q8TC27) expressed in HEK293 cells, which supports mammalian-relevant propeptide processing and disulfide folding. The prodomain is retained or cleaved depending on intracellular furin-like proprotein convertase activity in HEK293 — in practice you may observe a mixture of pro and processed forms, reflected as a minor doublet or broad band near 100–115 kDa. The lot-specific SDS-PAGE image in the CoA identifies the predominant species. If you require a defined propeptide-free ectodomain, contact technical support for available truncation variants.

How should I store and handle recombinant ADAM-32 to maintain stability after receipt?

Store at -20°C immediately upon receipt in single-use aliquots to avoid repeated freeze-thaw cycles, which accelerate aggregation of glycosylated ADAM ectodomains. Working aliquots can be held at 4°C for up to 72 hours; do not leave on ice for extended periods if CaCl2 is present in the buffer, as trace metal precipitation can occur. When diluting for assays, use the supplied buffer or PBS + 0.1% BSA as a carrier to minimize surface adsorption losses at low protein concentrations (<10 µg/mL). Centrifuge briefly at 10,000 × g before use if any visible particulate is present.