ADAM-20 (Recombinant)

Frequently Asked Questions

What molecular weight band should I expect for recombinant ADAM-20 on SDS-PAGE or Western blot?

The unprocessed ADAM-20 polypeptide (UniProt O43506) runs at a predicted ~83 kDa based on 726 amino acids, but the HEK293-expressed recombinant carries N-linked glycosylation characteristic of native ADAM ectodomains, so the apparent MW on reducing SDS-PAGE typically shifts to approximately 90–100 kDa. Under non-reducing conditions, intramolecular disulfide bonds in the cysteine-rich and disintegrin domains can cause anomalous migration. Always confirm your positive-control band position against a prestained ladder in the 90–110 kDa range when using this recombinant.

Is this recombinant ADAM-20 the full-length protein or a processed ectodomain fragment?

This recombinant corresponds to the ADAM-20 ectodomain — the prodomain through the EGF-like domain — expressed in HEK293 cells and secreted into conditioned medium. The transmembrane segment and cytoplasmic tail are excluded to permit secretion and purification. The prodomain may be auto-processed during expression, yielding the mature, active metalloproteinase form; lot-specific processing status is documented in each Certificate of Analysis. If your application requires the cytoplasmic tail epitope, this construct is not appropriate.

What substrates can I use to measure ADAM-20 proteolytic activity in a fluorescence assay?

ADAM-20 is a zinc-dependent metalloproteinase (EC 3.4.24.-) with substrate preferences broadly similar to other ADAM family members. A quenched fluorescent peptide such as Mca-KPLGL-Dpa-AR-NH2 or the generic ADAM substrate Mca-PLAQAV-Dpa-RSSSR-NH2 can serve as starting points for activity assays. Reactions should be run at 37 °C in assay buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, and 5 mM CaCl2 — matching the storage buffer to minimize dilution artifacts. Confirm substrate turnover with a metalloproteinase inhibitor control such as 10 µM GM6001.

What buffer conditions are optimal for ADAM-20 recombinant activity assays and should I add extra calcium?

The storage buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl2 — is formulated specifically for metalloproteinases and is compatible with direct use in activity assays after dilution. Glycerol carried over from the stock is generally tolerable at final concentrations below 1%. Do not reduce CaCl2 below 1 mM in the assay; calcium is required for metalloproteinase domain stability. Avoid EDTA or EGTA-containing buffers, which will chelate zinc and calcium and abolish activity. Zinc supplementation (1–10 µM ZnCl2) can improve activity if your diluent is metal-depleted.

What starting concentration of recombinant ADAM-20 should I use for an enzyme activity titration experiment?

For an initial activity titration, a useful starting range is 50–500 ng/mL (roughly 0.5–5 nM based on ~100 kDa apparent MW) in assay buffer with a fluorogenic peptide substrate at 10–50 µM. Titrate enzyme concentration while holding substrate constant to confirm linearity of signal over the first 30–60 minutes. At concentrations above 1 µg/mL, substrate depletion can become significant with sensitive peptides. For inhibitor IC50 determinations, fix enzyme at a concentration that gives 20–30% substrate conversion per hour to maintain assay sensitivity and avoid tight-binding artifacts.

Can I use recombinant ADAM-20 as a positive control for Western blot with the RP-ADAM20 rabbit polyclonal antibody?

Yes — this is one of the primary intended uses for REC-ADAM20. The matched antibody RP-ADAM20 (see /anti-adam-20-rabbit-polyclonal-antibody) was raised and validated against the same ADAM-20 ectodomain represented in this recombinant, so band recognition is assured. Load 20–50 ng of recombinant protein per lane alongside your cell or tissue lysates; this amount gives a clean, non-saturated signal at typical RP-ADAM20 dilutions. Using the recombinant as a positive control simultaneously confirms antibody performance and establishes the expected migration position in your specific gel system.

How much recombinant ADAM-20 should I load for a Western blot positive control and what antibody dilution works best?

Load 20–50 ng of REC-ADAM20 per lane for a standard positive control. At this amount, the RP-ADAM20 rabbit polyclonal antibody (SKU: RP-ADAM20) reliably detects a band in the 90–100 kDa region under standard reducing SDS-PAGE conditions. A primary antibody dilution of 1:500–1:2000 in 5% non-fat milk or BSA in TBST is a practical starting range; optimize based on your detection system and secondary antibody. ECL detection on a 10–12% polyacrylamide gel generally provides the cleanest resolution in this MW window.

How should I store recombinant ADAM-20 after receipt and how long is it stable once thawed?

Upon receipt, transfer REC-ADAM20 immediately to -20 °C. The product is supplied in single-use aliquots to eliminate repeated freeze-thaw cycles, which degrade metalloproteinase activity by disrupting zinc coordination in the catalytic domain. Once thawed, keep on ice and use within 4–8 hours; do not refreeze. For dilutions prepared in assay buffer, stability drops more rapidly — prepare working dilutions fresh on the day of use. Under recommended storage conditions, activity is maintained for at least 12 months from the date of manufacture, as stated on the Certificate of Analysis.