ACE-2 (Recombinant)

Frequently Asked Questions

What molecular weight band should I expect for recombinant ACE-2 on SDS-PAGE or Western blot?

The recombinant ACE-2 ectodomain (residues 18–740) has a predicted molecular weight of approximately 85 kDa based on amino acid sequence alone. However, because this protein is produced in HEK293 cells, N-linked glycosylation adds substantial mass — expect a diffuse band running between 100–120 kDa under denaturing conditions. The smearing is characteristic of native-like glycosylation and is not a sign of degradation. If you require a sharper band for a loading standard, PNGase F treatment will collapse the band toward the predicted 85 kDa backbone.

Which isoform or processing form of ACE-2 is in the Triple Point Biologics recombinant — full-length or ectodomain?

This product contains the soluble ectodomain corresponding to approximately residues 18–740 of the full-length 805-amino acid human ACE-2 (UniProt Q9BYF1). The N-terminal signal peptide (residues 1–17) and the single-pass transmembrane anchor plus cytoplasmic tail (roughly residues 741–805) are absent. This is the form released naturally by ADAM17/TACE shedding and is the biochemically active construct used in the vast majority of carboxypeptidase activity and receptor-binding studies.

What substrates does recombinant ACE-2 cleave and how do I measure carboxypeptidase activity in vitro?

ACE-2 cleaves a single C-terminal amino acid from its substrates. For fluorometric activity assays, the synthetic quenched substrate Mca-APK(Dnp) (also sold as ACE-2 Fluorogenic Substrate I) is the most commonly used option, generating a signal at Ex/Em 320/405 nm. Physiological substrates include angiotensin II (→ angiotensin 1-7), angiotensin I (→ angiotensin 1-9), apelin-13, apelin-17, and dynorphin A. Run reactions in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 µM ZnCl₂ at 37°C. Include 50 µM MLN-4760 or DX600 as inhibitor controls to confirm ACE-2-specific signal.

What starting concentration of recombinant ACE-2 should I use in a carboxypeptidase activity assay?

For initial dose-response or inhibitor IC50 experiments, 1–10 nM of this recombinant ACE-2 ectodomain is a practical starting range with the Mca-APK(Dnp) substrate at 10–50 µM. At 1 nM enzyme, you should achieve a linear signal increase for at least 30–60 minutes at 37°C, which is the window needed for reliable initial-velocity measurements. If your readout is endpoint rather than kinetic, 5 nM is a reasonable fixed concentration. Avoid exceeding 25 nM in inhibitor assays, as substrate depletion artifacts become significant within the assay window.

What buffer conditions are optimal for recombinant ACE-2 enzymatic activity, and does the storage buffer inhibit activity?

The storage buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol — is compatible with activity assays at dilutions of 1:10 or greater; glycerol carryover below 1% final concentration does not measurably suppress ACE-2 carboxypeptidase activity. ACE-2 is a zinc metalloproteinase, so avoid EDTA or EGTA in assay buffers, as chelation of the active-site Zn²⁺ causes rapid inactivation. Supplementing the assay buffer with 10 µM ZnCl₂ is recommended when working with dilute enzyme preparations or prolonged incubations. Optimal pH is 6.5–7.5; activity drops noticeably below pH 6.

Can I use Triple Point Biologics recombinant ACE-2 as a positive control for Western blot with the RP-ACE2 matched antibody?

Yes — this is one of the primary intended uses of REC-ACE2. The matched rabbit polyclonal antibody RP-ACE2 (/anti-ace-2-rabbit-polyclonal-antibody) is raised against the ACE-2 ectodomain and has been validated by Western blot against this exact recombinant protein. Load 25–100 ng of REC-ACE2 per lane alongside your cell lysate or tissue samples. Expect a band at 100–120 kDa due to glycosylation. Because both products come from the same laboratory, there is no ambiguity about epitope coverage or cross-reactivity — the pairing is experimentally confirmed, not predicted.

How much recombinant ACE-2 should I load per lane for Western blot positive control, and what dilution of RP-ACE2 antibody works?

Load 50 ng of REC-ACE2 per lane as a starting point; this reliably produces a strong, clean band at 100–120 kDa without saturating the signal. If you are titrating antibody concentration alongside cell lysates, a 25 ng lane for REC-ACE2 keeps the positive control from overwhelming adjacent sample lanes. For RP-ACE2, a primary antibody dilution of 1:500–1:2000 in 5% BSA/TBST is typical for Western blot; we recommend starting at 1:1000. These parameters were established with this recombinant as the standard, so signal and background should be predictable.

How should I handle, dilute, and store recombinant ACE-2 to maintain enzymatic activity long-term?

REC-ACE2 is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol and should be stored at -20°C in single-use aliquots immediately upon receipt. Avoid repeated freeze-thaw cycles — each cycle can reduce specific activity by 10–20% for metalloproteinases. For dilutions below 10 µg/mL, use assay buffer supplemented with 0.1% BSA as a carrier to prevent adsorptive loss to tube walls. Purity is >90% by SDS-PAGE and endotoxin is <0.1 EU/µg by LAL, so the preparation is suitable for cell-based assays where endotoxin contamination would confound results.