ADAM-21 (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant ADAM-21 on SDS-PAGE or Western blot?

The full-length human ADAM-21 protein (UniProt Q9UKJ8) has a calculated molecular weight of approximately 82 kDa based on its 722-amino-acid sequence. Because this recombinant is expressed in HEK293 cells, N-linked glycosylation is expected to shift the apparent MW on SDS-PAGE to roughly 90–100 kDa under reducing conditions. The precise band position can vary slightly by gel system and percentage. If you are running a Western blot positive control alongside our matched antibody RP-ADAM21, load 50–100 ng per lane and expect a diffuse band in the 90–100 kDa range consistent with glycoprotein migration.

Which processing form of ADAM-21 is present in this recombinant — full-length, prodomain-cleaved, or ectodomain only?

This recombinant corresponds to the mature, prodomain-processed ectodomain of human ADAM-21, encompassing the metalloproteinase, disintegrin, and cysteine-rich domains. The signal peptide and propeptide are removed during HEK293 expression and secretory processing, yielding an active enzyme fragment rather than the full 722-residue precursor. The transmembrane anchor is not included, which allows purification from the conditioned medium. This soluble, processed form is the relevant species for most in vitro metalloproteinase activity and binding assays.

What substrates can I use in an ADAM-21 metalloproteinase activity assay?

ADAM-21 is a zinc-dependent metalloproteinase (EC 3.4.24.-) and can be assayed using generic ADAM-family fluorogenic peptide substrates such as Mca-PLAQAV-Dpa-RSSSR-NH2 or related quenched fluorescent peptides based on the consensus ADAM cleavage motif. Monitor fluorescence at Ex/Em 320/405 nm. Because ADAM-21's specific endogenous substrates in vivo remain incompletely characterized, fluorogenic peptide substrates provide a practical proxy for measuring catalytic competence. Include a broad-spectrum metalloproteinase inhibitor such as GM6001 (10 µM) as a negative control to confirm that signal is zinc-dependent and proteinase-specific.

What buffer conditions are recommended for ADAM-21 activity assays, and does it need calcium?

The recombinant is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl2 — the calcium is included because ADAM family metalloproteinases require Ca²⁺ for structural stability of the catalytic domain. For activity assays, dilute into a reaction buffer of 25–50 mM Tris-HCl pH 7.4–7.5, 150 mM NaCl, 5–10 mM CaCl2, 0.05% Brij-35. Omitting calcium markedly reduces catalytic activity. Keep assay pH between 7.0 and 7.8; activity drops sharply outside this range. Pre-equilibrate protein and substrate to 37°C before combining to minimize lag-phase variability.

What starting enzyme concentration should I use for an ADAM-21 fluorogenic peptide activity assay?

A practical starting point is 1–5 nM recombinant ADAM-21 per reaction, with fluorogenic substrate at 10–20 µM. At these concentrations, substrate is well below saturation (Km for ADAM-family peptide substrates typically falls in the 5–50 µM range), keeping conditions in the linear, kinetically meaningful regime. If you are running inhibitor IC50 experiments, fix enzyme concentration at 2 nM, titrate inhibitor across at least eight concentrations spanning three orders of magnitude, and allow 10 minutes of enzyme-inhibitor pre-incubation at 37°C before adding substrate. Verify linearity of the fluorescence signal over at least 30 minutes before calculating initial velocity.

Can I use recombinant ADAM-21 as a positive control on a Western blot when validating the RP-ADAM21 antibody?

Yes — this is a primary use case. Load 50–100 ng of recombinant ADAM-21 (REC-ADAM21) per lane alongside your cell lysate samples. After transfer and blocking, use the matched rabbit polyclonal antibody RP-ADAM21 (see /anti-adam-21-rabbit-polyclonal-antibody) at a recommended starting dilution of 1:500–1:1000 in 5% non-fat milk/TBST. Expect a band at approximately 90–100 kDa for the recombinant and a band at 90–100 kDa (or higher if full-length precursor is present) in ADAM-21-expressing cell lysates. The recombinant and antibody are produced and validated in the same laboratory, eliminating the guesswork around epitope compatibility.

How much recombinant ADAM-21 should I load for a Western blot positive control, and will it over-saturate the blot?

50 ng is sufficient for a clean, non-saturating positive control band with the RP-ADAM21 antibody under standard ECL detection. If you are using a high-sensitivity HRP substrate (e.g., femtogram-range detection systems), reduce loading to 10–25 ng to avoid blooming. Because the recombinant is >90% pure by SDS-PAGE, background bands are minimal and will not confound interpretation. Run it in a lane adjacent to — not co-mingled with — your experimental lysates so that antibody titration across the blot remains consistent. A 10% SDS-PAGE gel resolves the ~90–100 kDa band cleanly away from common contaminants.

How should I store and handle recombinant ADAM-21 to maintain activity, and what is its shelf life?

Store at −20°C in the single-use aliquots provided. The storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl2) is formulated to stabilize the metalloproteinase domain through freeze-thaw. Repeated freeze-thaw cycles progressively reduce activity — once an aliquot is thawed, keep it on ice and use within 4–6 hours. Shelf life is 12 months from the date of receipt when stored correctly. For dilutions below 10 µg/mL, add carrier protein (0.1% BSA, protease-free) to prevent surface adsorption losses. Do not add EDTA or EGTA to working buffers; chelation of Zn²⁺ and Ca²⁺ will inactivate the enzyme irreversibly.