ADAM-18 (Recombinant)

Frequently Asked Questions

What molecular weight band should I expect for recombinant ADAM-18 on SDS-PAGE or Western blot?

The full-length human ADAM-18 sequence (UniProt Q9Y3Q7) encodes 739 amino acids, giving a theoretical unglycosylated MW of approximately 83 kDa. Because this recombinant is expressed in HEK293 cells, native-like N-glycosylation is retained, and the observed band on reducing SDS-PAGE typically runs between 95–110 kDa. The diffuse or slightly smeary appearance is consistent with heterogeneous glycosylation, which is expected for a sperm-surface glycoprotein. If you are using this as a positive control alongside our matched antibody RP-ADAM18, load 50–100 ng per lane for a clean, unambiguous signal.

Is recombinant ADAM-18 proteolytically active, and what substrate should I use to measure activity?

ADAM-18 is annotated as a non-catalytic metalloprotease-like protein. Its metalloproteinase domain lacks the conserved HExxHxxGxxH zinc-coordinating residues required for catalytic activity, so ADAM-18 does not cleave canonical ADAM fluorogenic substrates such as Mca-KPLGL-Dpa-AR-NH2. Do not design activity assays premised on proteolytic cleavage. Research applications are better oriented toward binding interaction studies — using the disintegrin-like and cysteine-rich domains — rather than enzyme kinetics. Plan accordingly when selecting positive controls for metalloprotease activity panels.

What buffer is recombinant ADAM-18 supplied in, and does it interfere with downstream binding assays?

This recombinant is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, with 5 mM CaCl2 included consistent with our metalloproteinase-family formulation. For surface plasmon resonance or biolayer interferometry binding assays, the glycerol and CaCl2 are generally compatible at working dilutions (typically 1:10–1:50 into your running buffer). If your assay is calcium-sensitive or requires EDTA-based buffers, pre-dialyze the protein against a compatible buffer using a 10 kDa MWCO membrane. Avoid EDTA entirely, as it may affect domain conformation even in the absence of catalytic activity.

What starting concentration of recombinant ADAM-18 should I use for protein interaction or pull-down experiments?

For pull-down or co-immunoprecipitation assays probing ADAM-18's disintegrin-like or cysteine-rich domain interactions, a starting bait concentration of 1–5 µg per reaction in standard co-IP lysis buffer (supplemented with 1 mM CaCl2 to maintain structural integrity) is a practical starting point. Titrate prey protein at 2- to 5-fold molar excess over ADAM-18 to favor complex formation. Because ADAM-18 has no catalytic activity, no protease inhibitor cocktail is needed to preserve the bait, though including it will protect endogenous prey proteins from degradation during incubation.

Can I use recombinant ADAM-18 as a positive control for Western blot with the RP-ADAM18 rabbit polyclonal antibody?

Yes — this is one of the primary validated use cases. REC-ADAM18 and RP-ADAM18 are produced and validated together in the same lab, so compatibility for Western blot is confirmed rather than inferred. Load 50–100 ng of REC-ADAM18 per lane under reducing conditions; expect a band at 95–110 kDa due to glycosylation. Use RP-ADAM18 at 1:500–1:2,000 dilution with standard HRP-conjugated secondary. This setup gives you an unambiguous reference band alongside your cell lysate lanes and is directly useful for establishing antibody linearity and lot-to-lot consistency.

How do I use recombinant ADAM-18 to validate the RP-ADAM18 antibody for immunohistochemistry?

For IHC antibody validation, spike REC-ADAM18 into a carrier protein matrix (e.g., 1% BSA in PBS) at 0.5–2 µg, spot or embed into an agar cell block, and section alongside your tissue of interest. RP-ADAM18 is predicted to recognize denatured epitopes given its polyclonal nature, but confirm antigen retrieval conditions (citrate pH 6.0 is a standard first attempt) using the recombinant-spiked control before committing tissue sections. This approach lets you decouple antibody sensitivity from endogenous expression variability before interpreting tissue staining patterns.

How should I store recombinant ADAM-18 and what is its expected shelf life after thawing?

Store REC-ADAM18 at -20°C in the single-use aliquots as supplied. The 10% glycerol in the buffer provides cryoprotection without requiring liquid nitrogen storage. Shelf life is 12 months from receipt at -20°C, provided freeze-thaw cycles are avoided — each cycle measurably reduces recovery of properly folded protein, particularly for the disulfide-rich cysteine-rich domain. Once thawed, keep on ice and use within 4–8 hours. Do not refreeze. If your experiment requires multiple small doses, request custom aliquot sizes at time of order or aliquot yourself immediately upon first thaw using a low-protein-binding tube.

Does recombinant ADAM-18 include the prodomain, and what processing form is represented?

This recombinant represents the mature ectodomain form of human ADAM-18, with the signal peptide and prodomain removed consistent with post-translational processing by furin-like proprotein convertases that occurs for ADAM family members. The expressed construct spans the metalloproteinase-like, disintegrin-like, cysteine-rich, and EGF-like domains but excludes the transmembrane and cytoplasmic regions, making it soluble and directly suitable for extracellular interaction studies. If your experimental model requires the full transmembrane context, cell-surface expression systems should be considered rather than soluble recombinant protein.