ACE-1 (Recombinant)

Frequently Asked Questions

What molecular weight should I expect for ACE-1 (Recombinant) on SDS-PAGE or Western blot?

The recombinant ACE-1 ectodomain runs at approximately 140–170 kDa under reducing SDS-PAGE conditions. The predicted MW of the mature ectodomain is ~140 kDa, but extensive N-linked glycosylation — a key reason we selected HEK293 expression — broadens the band and shifts apparent MW upward. Purity is >95% at this band by Coomassie staining. If you are running a deglycosylation experiment (e.g., PNGase F treatment), expect a shift down to ~120 kDa, consistent with the protein backbone alone.

What is the exact form of ACE-1 (Recombinant) — full-length, ectodomain, or a single catalytic domain?

This product is the soluble ectodomain of human ACE (UniProt P12821), corresponding to the physiologically shed form known as CD143 detected in plasma. It retains both the N-domain and C-domain, each with an intact HEXXH zinc-coordinating motif, so you can study domain-selective substrate preferences or inhibitor binding without needing to express the domains separately. The single-pass transmembrane anchor and cytoplasmic tail are absent, which is consistent with the soluble, active form isolated from human plasma in vivo.

What substrates and assay conditions work best for measuring ACE-1 activity in vitro?

The most widely used fluorogenic substrate is Abz-FRK(Dnp)P-OH or the chromogenic substrate Hip-His-Leu (hippuryl-histidyl-leucine), hydrolyzed at the His-Leu bond to release hippuric acid (monitored at 228 nm). For fluorogenic assays, Mca-RPPGFSAFK(Dnp)-OH is also well-validated. Use our standard activity buffer: 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 µM ZnCl₂, 5 mM CaCl₂. Run reactions at 37°C. A starting enzyme concentration of 1–5 nM is appropriate for initial velocity measurements; titrate substrate from 50 µM to 1 mM to determine Km under your specific conditions.

How do I optimize buffer conditions for ACE-1 (Recombinant) activity assays, and does EDTA interfere?

ACE-1 is a zinc metallopeptidase and is acutely sensitive to metal chelators. Even 0.1 mM EDTA or EGTA will substantially reduce activity by stripping the catalytic Zn²⁺; avoid both in assay buffers. The product is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl₂. For activity assays, dilute directly into the same buffer minus glycerol, supplemented with 10 µM ZnCl₂. Optimal pH is 7.0–8.0; activity drops noticeably below pH 6.5. Chloride ion concentration also modulates domain selectivity — higher [Cl⁻] (>100 mM) preferentially activates the C-domain.

What starting concentration of ACE-1 (Recombinant) should I use for IC50 inhibitor profiling with captopril or lisinopril?

For IC50 determinations, 1–2 nM enzyme is standard with a fluorogenic substrate (e.g., Mca-RPPGFSAFK(Dnp)-OH) at a concentration near the apparent Km (~100–200 µM depending on domain). Captopril has a reported IC50 in the sub-nanomolar range (~1–5 nM) against the C-domain, so ensure your enzyme concentration is well below inhibitor Ki to avoid stoichiometric binding artifacts. Lisinopril inhibits both domains with similar potency. Include 0.1% BSA in dilution buffer to minimize enzyme adsorption to plate surfaces at these low concentrations.

Can I use ACE-1 (Recombinant) as a positive control for Western blot with the matched antibody RP-ACE1?

Yes — that is the primary design intent of this pairing. Load 20–50 ng of ACE-1 (Recombinant) per lane alongside your cell lysate samples. At this amount, RP-ACE1 (rabbit polyclonal; /anti-ace-1-rabbit-polyclonal-antibody) reliably detects the glycosylated ectodomain band at ~140–170 kDa. Because RP-ACE1 and REC-ACE1 are produced and validated in the same lab, you avoid the common problem of a recombinant and antibody sourced from different vendors recognizing different epitopes or denatured vs. native forms inconsistently.

How much recombinant ACE-1 (Recombinant) should I load for Western blot, and will it run at the same size as endogenous ACE?

Load 20–50 ng for a clean single-band signal with RP-ACE1 under standard ECL detection. The recombinant ectodomain will run at ~140–170 kDa — overlapping with endogenous, membrane-shed ACE detected in cell lysates expressing the full-length protein. If you need to distinguish them, note that the full-length transmembrane form runs slightly higher (~180 kDa) due to the intact stalk and transmembrane region. The recombinant serves as an unambiguous positive control band to confirm antibody functionality before interpreting endogenous signal in experimental lanes.

How should I store and handle ACE-1 (Recombinant) to preserve activity, and what is its shelf life after thawing?

Store at -20°C in the supplied single-use aliquots (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl₂). Avoid repeated freeze-thaw cycles — each cycle measurably reduces metallopeptidase activity. Once thawed, keep on ice and use within 4–6 hours; do not refreeze. For dilutions below ~10 nM, add 0.1% BSA (protease-free) to prevent surface adsorption losses. Shelf life is 12 months from date of manufacture when stored correctly at -20°C. Endotoxin is confirmed <0.1 EU/µg by LAL, so the preparation is suitable for cell-based assays without additional depyrogenation steps.