Carboxypeptidase A-2 (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant CPA2 on SDS-PAGE and Western blot?

The CPA2 precursor is 419 amino acids (~47 kDa predicted), but the HEK293-expressed recombinant typically migrates at approximately 50–55 kDa under reducing SDS-PAGE conditions due to N-linked glycosylation acquired in the mammalian expression system. The propeptide may be partially retained depending on lot processing; consult the certificate of analysis for confirmation of the active vs. zymogen form. On Western blot using our matched antibody (RP-CarboxypeptidaseA2), a clean band in the 50–55 kDa range is expected with minimal background at recommended dilutions.

Does this recombinant CPA2 include the propeptide, or is it the mature active form?

This recombinant is produced as the mature, activated form — the propeptide segment responsible for latency in the zymogen has been removed during processing. The native CPA2 zymogen is activated by tryptic cleavage of the ~10 kDa pro-domain; the recombinant supplied here reflects the extracellular active enzyme state. If you require the pro-form for activation kinetic studies, this product is not appropriate for that application. For active-site titration or substrate cleavage assays, the processed form is ready to use without additional activation steps.

What substrates are recommended for a CPA2 activity assay, and how does selectivity differ from CPA1?

CPA2 shows pronounced selectivity for bulky aromatic C-terminal residues — substrates such as N-(4-methoxyphenylazobenzyloxycarbonyl)-Phe (AAFP) and hippuryl-Phe (Hip-Phe) are well-characterized chromogenic substrates. CPA1, by contrast, preferentially cleaves aliphatic residues. For fluorometric assays, Hip-Phe at 0.5–2 mM in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl₂ at 37°C is a practical starting point. Confirm linearity of signal with respect to enzyme concentration before IC₅₀ determinations to avoid substrate depletion artefacts.

What buffer conditions work best for CPA2 activity assays with this recombinant?

The storage buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl₂ — is compatible with most activity assay formats without dilution-induced inactivation at working concentrations above 1 nM. Calcium is essential for structural stability; do not use EDTA or EGTA in assay buffers, as zinc and calcium chelation will inactivate the enzyme. If your assay requires a phosphate-based buffer, validate activity retention first. Glycerol from the storage buffer at final dilutions below 0.5% does not typically inhibit activity, but run a glycerol-only blank if you observe baseline drift.

What starting concentration of recombinant CPA2 should I use in a kinetic inhibition or IC50 assay?

A starting concentration of 1–5 nM recombinant CPA2 is appropriate for most Michaelis-Menten and inhibition assays using chromogenic or fluorogenic C-terminal substrates. At this range the enzyme is well below substrate saturation with typical 0.5–2 mM substrate concentrations, keeping conditions within the linear velocity regime. For tight-binding inhibitors (Ki below ~10× enzyme concentration), apply Morrison's equation rather than classical IC₅₀ fitting. Titrate enzyme concentration independently against your substrate batch, as chromogenic substrate purity varies between suppliers and affects apparent Km.

Can I use recombinant CPA2 as a positive control on Western blot with the RP-CarboxypeptidaseA2 antibody?

Yes — this recombinant is the recommended positive control for RP-CarboxypeptidaseA2 (our matched rabbit polyclonal, available at /anti-carboxypeptidase-a2-rabbit-polyclonal-antibody). The antibody was raised and validated against the same protein, guaranteeing signal detection. Load 20–50 ng of recombinant CPA2 per lane alongside your cell lysate or tissue samples. Expect a band at ~50–55 kDa. The recombinant-antibody pair is particularly useful for confirming antibody lot-to-lot consistency and for calibrating signal in quantitative Western blot workflows.

How much recombinant CPA2 should I load for a Western blot positive control lane?

Load 20–50 ng per lane as a starting point when using RP-CarboxypeptidaseA2 at its recommended dilution (typically 1:500–1:2000; verify on the antibody datasheet). At 20 ng you should observe a clear, unsaturated band at ~50–55 kDa that does not bleed into adjacent lanes. If you are running a quantitative densitometry experiment, a two-point standard curve at 10 ng and 50 ng alongside lysate allows approximate normalization. Reduce load to 5–10 ng if using enhanced chemiluminescence substrates with long exposure times to avoid signal saturation.

How should I handle, dilute, and store recombinant CPA2 to maintain activity over time?

Aliquot immediately upon receipt and store at −20°C; avoid repeated freeze-thaw cycles, which progressively denature the zinc-metallocarboxypeptidase active site. Each aliquot should be thawed once, used, and discarded. For working dilutions, use the storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl₂, 10% glycerol) or supplement your assay buffer with 0.1% BSA as a carrier to limit adsorptive losses at sub-nanomolar concentrations. At −20°C in the supplied buffer, activity is stable for at least 12 months from the date of manufacture per internal stability data.