ADAM-15 (Recombinant)

Frequently Asked Questions

What molecular weight band should I expect for recombinant ADAM-15 on SDS-PAGE or Western blot?

The full-length human ADAM-15 polypeptide encodes 863 amino acids, giving a predicted unglycosylated MW of ~98 kDa. Because this preparation is produced in HEK293 cells with native-like N-glycosylation, the observed band on reducing SDS-PAGE typically runs at 100–115 kDa depending on glycan load and the expressed region in your specific lot. Always confirm the precise expressed region and tag contribution from your current lot datasheet. Under non-reducing conditions, apparent MW may shift further due to intramolecular disulfide architecture in the cysteine-rich domain.

Is the recombinant ADAM-15 the pro-domain form or the mature processed form?

ADAM-15 undergoes autocatalytic or furin-mediated removal of its N-terminal pro-domain as part of activation. The HEK293 expression system used here supports endogenous proprotein convertase activity, so a significant proportion of the preparation is in the mature, pro-domain-cleaved form. However, a mixture of precursor and processed species is common with ADAM family members. Your lot datasheet will indicate the dominant species observed by SDS-PAGE. If you need to confirm activation state, the metalloproteinase inhibitor EDTA (5–10 mM) will suppress any residual auto-processing during analysis.

What substrates can I use to measure ADAM-15 metalloproteinase activity in a fluorescent assay?

ADAM-15 cleaves peptide substrates containing the core sequence recognized by meltrin-family sheddases. Internally quenched fluorescent (IQF) substrates such as Mca-KPLGL-Dpa-AR-NH2 or the generic ADAM substrate Mca-PLAQAV-Dpa-NH2 are commonly used starting points. Assay in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl2 (matching the storage buffer) at 37°C. A starting enzyme concentration of 50–200 nM with substrate at 10–20 µM provides a linear initial velocity window. Include 10 mM EDTA or 10 µM GM6001 as a negative control to confirm metal-dependent activity.

What buffer conditions are optimal for ADAM-15 activity assays and does the storage buffer interfere?

The storage buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl2 — is directly compatible with standard ADAM-15 activity assays. Dilute the enzyme at least 1:10 into assay buffer to bring glycerol to ≤1%, which minimizes viscosity effects on fluorescence readings. Maintain 1–5 mM CaCl2 in the final assay; calcium chelation by residual EDTA from other reagents is a common cause of activity loss. Avoid Zn²⁺-chelating buffers (e.g., HEPES with trace EDTA) without supplementing ZnCl2 at 1–10 µM to support the catalytic zinc center.

What starting concentration of recombinant ADAM-15 should I use for an inhibitor IC50 experiment?

For IC50 determination, use 25–100 nM recombinant ADAM-15 with your IQF substrate at a concentration at or below the apparent Km (typically 10–20 µM for generic ADAM substrates). Keeping enzyme well below substrate concentration ensures you are measuring inhibitor competition under conditions approximating Cheng-Prusoff validity. Pre-incubate enzyme with inhibitor for 15–30 minutes at room temperature before adding substrate to allow equilibrium binding, particularly for slow-on inhibitors like hydroxamic acid-based compounds. Run each IC50 curve with a GM6001 reference standard to confirm assay consistency between experiments and lots.

Can I use recombinant ADAM-15 as a positive control for Western blot with the matched TPB antibody RP-ADAM15?

Yes — this is the most straightforward use case. RP-ADAM15 (/anti-adam-15-rabbit-polyclonal-antibody) is produced in the same laboratory against ADAM-15 antigen and is guaranteed compatible with this recombinant for Western blot. Load 20–50 ng of recombinant ADAM-15 per lane on a 4–12% Bis-Tris gel; expect a band at 100–115 kDa. This loading is sufficient for clear detection with RP-ADAM15 at standard dilutions without saturating the signal, leaving room to compare against endogenous ADAM-15 in your cell lysate lanes on the same membrane.

How much recombinant ADAM-15 should I load for a Western blot positive control and what dilution of RP-ADAM15 antibody should I use?

Load 20–50 ng of recombinant ADAM-15 per lane. At this quantity, RP-ADAM15 (SKU: RP-ADAM15) used at 1:500–1:2000 in 5% non-fat milk or BSA in TBST will produce a clean band at 100–115 kDa within standard ECL exposure times (1–5 minutes). If you are co-detecting endogenous ADAM-15 in tissue lysates — which express the protein at lower levels — titrate the antibody toward 1:500 and increase lysate loading to 20–30 µg total protein. The recombinant positive control lane anchors band identity and confirms antibody lot-to-lot consistency.

How should I handle, dilute, and store recombinant ADAM-15 to maintain activity over time?

Recombinant ADAM-15 is supplied as a sterile solution in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl2. Upon receipt, aliquot into single-use volumes before freezing at -20°C; repeated freeze-thaw cycles accelerate aggregation and loss of metalloproteinase activity. For working dilutions, use the storage buffer or a compatible assay buffer containing 0.1% BSA as a carrier when diluting below ~100 ng/µL to prevent adsorptive losses to tube walls. Diluted enzyme should be used within 4–8 hours on ice. Do not store diluted working stocks.