ADAM-28 (Recombinant)

Frequently Asked Questions

What molecular weight band should I expect for recombinant ADAM-28 on SDS-PAGE or Western blot?

The full-length human ADAM-28 protein has a predicted molecular weight of ~88 kDa from sequence alone, but the HEK293-expressed recombinant typically migrates at approximately 90–100 kDa under reducing SDS-PAGE conditions due to mammalian N-linked glycosylation. The prodomain is retained in some preparations; if your lot is the processed, prodomain-cleaved form, you may observe a band closer to 65–70 kDa corresponding to the mature metalloprotease/disintegrin fragment. Check your Certificate of Analysis for the specific processed state of your lot. Our matched antibody RP-ADAM28 resolves both forms cleanly on Western blot.

Is Triple Point Biologics recombinant ADAM-28 the full-length form or the secreted ADAM-28s splice variant?

This product (REC-ADAM28) is produced as the catalytic ectodomain fragment encompassing the prodomain through the cysteine-rich region, which corresponds functionally to the secreted ADAM28s splice variant that is detectable in plasma and lymphocyte supernatants. It does not include the transmembrane or cytoplasmic tail. This makes it directly comparable to the secreted isoform used in substrate-cleavage and shedding assays. If your experiment requires the membrane-anchored context, a cell-overexpression model would be more appropriate than this recombinant.

What substrates does recombinant ADAM-28 cleave and how do I set up an activity assay?

ADAM-28 cleaves myelin basic protein (MBP) and has well-documented sheddase activity toward IGFBP-3 and VLA-4 (α4-integrin ectodomain). For a fluorogenic activity assay, a generic ADAM substrate such as Mca-KPLGL-Dpa-AR-NH2 is functional, though ADAM-28 shows relatively modest activity on pan-ADAM peptides compared to ADAM-10/17. A practical starting condition: 200–500 ng recombinant ADAM-28 per 50 µL reaction in 25 mM Tris-HCl pH 7.5, 5 mM CaCl2, 0.1% Brij-35 at 37°C, reading fluorescence at Ex/Em 320/405 nm. Include 10 mM EDTA as a negative control to confirm metal-dependence.

What buffer is recombinant ADAM-28 supplied in and does it interfere with downstream enzymatic assays?

REC-ADAM28 is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl2. The glycerol stabilises the metalloprotease fold during freeze-thaw but should be diluted to ≤1% in activity assays to avoid viscosity-related artifacts. The 5 mM CaCl2 is compatible with standard ADAM assay buffers; no calcium chelation step is needed. If your assay buffer uses HEPES instead of Tris, a 1:10–1:20 dilution into HEPES-based assay buffer is generally sufficient to reduce Tris carry-over below interference thresholds. Avoid adding EDTA or EGTA to working dilutions, as these will chelate the catalytic zinc.

What starting concentration of recombinant ADAM-28 should I use for IC50 inhibitor screening?

For inhibitor IC50 determination against a fluorogenic substrate, we recommend titrating REC-ADAM28 between 50–200 ng per 50 µL reaction to identify a concentration that produces a linear signal increase over 30–60 minutes, staying well below substrate depletion. Using the lowest enzyme concentration that gives a robust signal improves sensitivity for tight-binding inhibitors (Ki < 10 nM). Broad-spectrum metalloprotease inhibitors such as GM6001 (10 µM) serve as a reliable positive inhibition control. Pre-incubate enzyme with inhibitor for 15 minutes at room temperature before adding substrate.

Can I use Triple Point Biologics recombinant ADAM-28 as a Western blot positive control with the matched RP-ADAM28 antibody?

Yes — this is one of the primary validated uses of REC-ADAM28. The matched rabbit polyclonal RP-ADAM28 (/anti-adam-28-rabbit-polyclonal-antibody) was raised and validated in the same laboratory and is guaranteed compatible with this recombinant for Western blot. Load 10–50 ng of REC-ADAM28 per lane alongside your cell lysate samples. This loading range produces a strong, clean band that confirms antibody performance and molecular weight calibration in the same blot, without overloading lanes adjacent to lower-expressing biological samples.

How much recombinant ADAM-28 should I load for a Western blot positive control and what band size should I expect?

Load 20–50 ng per lane as a positive control when using RP-ADAM28 at its recommended working dilution (typically 1:500–1:2000; confirm on the antibody datasheet). Under reducing conditions, expect the primary band at 90–100 kDa for the glycosylated ectodomain form. A faint lower band near 65–70 kDa may appear if partial prodomain processing occurred. Both bands are recognised by RP-ADAM28. Running this recombinant alongside MCF-7 or Raji cell lysates — cell lines with documented endogenous ADAM-28 expression — provides a useful side-by-side confirmation of endogenous versus recombinant protein migration.

How should I handle, dilute, and store recombinant ADAM-28 to maintain activity after receiving it?

Upon receipt, briefly centrifuge the vial and place immediately at -20°C. REC-ADAM28 is shipped as single-use aliquots to eliminate repeated freeze-thaw, which progressively denatures the metalloprotease active site. Avoid storing working dilutions at 4°C for more than 24 hours. For assay setup, thaw on ice, dilute into cold assay buffer containing 5 mM CaCl2, and use within 2 hours of thawing. Do not vortex; mix gently by pipetting. Adding 0.1% BSA (protease-free) to dilution buffer helps minimise adsorptive losses when working at concentrations below 50 ng/µL.