ADAM-11 (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant ADAM-11 on SDS-PAGE and Western blot?

The ADAM-11 open reading frame encodes a 769-amino-acid precursor with a calculated molecular weight of ~87 kDa. On reducing SDS-PAGE, our HEK293-expressed recombinant typically migrates at 95–110 kDa due to N-linked glycosylation, which is retained because of the mammalian expression system. Under non-reducing conditions, migration may shift slightly owing to intramolecular disulfide bonds in the cysteine-rich and disintegrin domains. Purity is >90% by SDS-PAGE. If your gel shows a diffuse band in this range, glycan heterogeneity is the most likely explanation — PNGase F treatment will sharpen the band and confirm identity.

Is recombinant ADAM-11 proteolytically active and what substrate should I use for activity assays?

ADAM-11 is a non-catalytic member of the ADAM family. Its metalloprotease-like domain lacks the canonical HExxHxxGxxH zinc-binding motif required for catalytic activity, so it does not cleave standard ADAM fluorogenic peptide substrates such as Mca-KPLGL-Dpa or TNF-α-derived quenched peptides. Do not use this recombinant in protease activity assays expecting ectodomain shedding. Its documented function is as a presynaptic cell-surface ligand — experiments are better directed toward integrin-binding assays, co-immunoprecipitation, or surface plasmon resonance to characterise protein–protein interactions.

What buffer is recombinant ADAM-11 supplied in and is it compatible with my binding or cell assay?

The recombinant is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol. Note that, unlike catalytically active ADAMs, the formulation does not include CaCl₂, because ADAM-11 has no active-site metal requirement. The 10% glycerol is a cryoprotectant; if glycerol interferes with your downstream assay (e.g., SPR, ITC, or cell-surface binding), buffer-exchange via a 10 kDa MWCO centrifugal filter into your assay buffer is straightforward. Keep the protein on ice during manipulation and avoid repeated freeze-thaw cycles from your single-use aliquots.

What starting concentration of recombinant ADAM-11 should I use for integrin-binding or pull-down experiments?

For solid-phase integrin-binding assays or GST pull-downs, a working range of 0.5–5 µg/mL ADAM-11 is a reasonable starting point, consistent with concentrations used for related non-catalytic ADAMs (e.g., ADAM-22, ADAM-23) in published binding studies. Titrate in at least three concentrations to establish a dose–response curve before committing to a fixed concentration. For co-immunoprecipitation from neuronal lysates, spike in 1–2 µg of recombinant per 500 µg total lysate as a positive control. Confirm capture using our matched antibody RP-ADAM11 on the Western blot.

Can I use recombinant ADAM-11 as a positive control for Western blot with the RP-ADAM11 antibody?

Yes — this is one of the primary intended uses of REC-ADAM11. The matched rabbit polyclonal RP-ADAM11 (/anti-adam-11-rabbit-polyclonal-antibody) was validated against this same recombinant, so band identity and migration are confirmed. Load 20–50 ng of REC-ADAM11 per lane alongside your experimental samples; this amount is well within the linear detection range of RP-ADAM11 on standard PVDF membranes. Expect a diffuse band at 95–110 kDa. This pairing is particularly useful when optimising antibody dilution or when running ADAM-11 Western blots on brain or cerebellar lysates where endogenous signal is low.

How much recombinant ADAM-11 should I load for a Western blot positive control, and at what antibody dilution?

Load 20–50 ng of REC-ADAM11 per lane. At this amount, RP-ADAM11 (SKU: RP-ADAM11) at 1:1,000–1:2,000 dilution in 5% non-fat milk/TBST reliably detects a clean band at 95–110 kDa within a standard 1-hour HRP incubation. If background is high in tissue lysate lanes, try 3% BSA/TBST for blocking — this is especially helpful with cerebellar or DRG lysates that are rich in endogenous IgG. The recombinant lane serves as both a migration marker and a sensitivity benchmark for each blot.

What is the isoform and processing state of the recombinant ADAM-11 — is it the full-length precursor or the mature ectodomain?

REC-ADAM11 is expressed from a construct encoding the full-length 769-amino-acid human ADAM-11 (UniProt O75078), including the prodomain, metalloprotease-like domain, disintegrin domain, cysteine-rich domain, and EGF-like domain, but excluding the single-pass transmembrane anchor and cytoplasmic tail, making it a soluble ectodomain fragment suitable for in vitro work. The prodomain may be partially auto-removed during HEK293 secretion, as seen with related ADAMs, resulting in a mixed population of pro- and mature-form protein. SDS-PAGE under reducing conditions will reveal whether prodomain cleavage is complete in your specific lot.

How should I store and handle recombinant ADAM-11 to maintain integrity over time?

Store at -20°C in the single-use aliquots provided. Avoid repeated freeze-thaw cycles — each cycle risks aggregation and loss of structural integrity of the disintegrin and cysteine-rich domains. Once thawed, keep on ice and use within 8 hours. If you need to dilute for assay, use a carrier protein such as 0.1% BSA in PBS or your assay buffer to prevent adsorption to tube walls at low concentrations (<10 µg/mL). Properly stored aliquots are stable for up to 12 months at -20°C. Do not store at -80°C long-term without first confirming stability in your specific matrix.