Carboxypeptidase A-1 (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant CPA1 on SDS-PAGE or Western blot?

The full-length human CPA1 precursor is 419 amino acids (~47 kDa predicted from sequence). Because this recombinant is expressed in HEK293 cells, N-linked glycosylation adds mass, so the apparent band on reducing SDS-PAGE typically runs between 50–55 kDa. The propeptide (~94 residues) may or may not be cleaved depending on the lot's processing state; check the Certificate of Analysis for the confirmed apparent MW of your specific lot. Purity is >95% by SDS-PAGE, so you should see a single predominant band with minimal higher-MW species.

Is the recombinant CPA1 supplied as the zymogen precursor or the processed active enzyme?

This product is supplied as an active enzyme form, not the full zymogen. The CPA1 precursor undergoes propeptide removal upon secretion; HEK293 expression recapitulates this secretion-coupled processing, yielding a form analogous to the mature secreted enzyme (approximately residues 95–419 of the UniProt P15085 canonical sequence). The active zinc-dependent metallocarboxypeptidase domain is intact and catalytically competent as supplied. If your experiment specifically requires the zymogen form, this product is not appropriate — contact us before ordering.

What substrates should I use to measure recombinant CPA1 carboxypeptidase activity in vitro?

The most widely used chromogenic substrate for CPA1 (EC 3.4.17.1) activity assays is hippuryl-L-phenylalanine (Hip-Phe), with absorbance monitored at 254 nm after hippuric acid release. Hippuryl-L-arginine is a useful negative-control substrate because CPA1 shows little activity toward C-terminal basic residues (Arg, Lys). Fluorogenic substrates such as Abz-Gly-Phe(NO2)-OH also work well for higher-sensitivity plate-based formats. Ensure your substrate solution is freshly prepared; carry the blank without enzyme to subtract non-enzymatic hydrolysis.

What buffer conditions are optimal for recombinant CPA1 activity assays?

CPA1 is a zinc-dependent metallocarboxypeptidase, so avoid chelating agents (EDTA, EGTA) entirely — they will strip the catalytic Zn²⁺ and abolish activity. The storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl₂) is compatible with most activity assays when diluted ≤1:10 into the reaction. Optimal pH is 7.5–8.0 for most hydrophobic C-terminal substrates. Including 0.01% BSA in the dilution buffer helps stabilize activity at low enzyme concentrations (<10 nM). Keep reactions at 37°C for physiological relevance.

What starting concentration of recombinant CPA1 should I use for a kinetic activity assay?

For initial Km/Vmax determination with Hip-Phe, a useful starting point is 5–20 nM CPA1 with substrate concentrations spanning 0.1–5 mM. At these enzyme concentrations the reaction should be linear for at least 10–15 minutes at 37°C. If signal is low, scale up to 50 nM; if background hydrolysis is masking the signal, reduce substrate concentration or drop enzyme to 2–5 nM. For inhibitor IC50 studies, titrate inhibitor against a fixed enzyme concentration producing ~20–30% substrate conversion at the assay endpoint to maintain sensitivity.

Can I use recombinant CPA1 as a positive control for Western blot with the matched RP-CarboxypeptidaseA1 antibody?

Yes — this is the primary intended cross-use case. The matched rabbit polyclonal antibody (SKU: RP-CarboxypeptidaseA1) was raised and validated in the same laboratory that produced this recombinant, so compatibility on Western blot is confirmed. Load 50–200 ng of recombinant CPA1 per lane alongside your cell lysate or tissue samples. The antibody detects the processed ~50–55 kDa HEK293-expressed band cleanly under denaturing conditions. This pairing is particularly useful when validating the antibody against low-expressing samples where an unambiguous positive control band is essential.

How much recombinant CPA1 should I load for a Western blot positive control lane?

100 ng per lane is a reliable starting point when using RP-CarboxypeptidaseA1 at a primary antibody dilution of 1:500–1:2000. If your detection system (ECL, fluorescent secondary) is highly sensitive, titrate down to 25–50 ng to avoid signal saturation that can obscure accurate band-size estimation. Boil the recombinant in 1× Laemmli buffer with β-mercaptoethanol for 5 minutes before loading — the glycosylated ~50–55 kDa band is fully detected under reducing SDS-PAGE conditions. Avoid loading >500 ng, as glycoprotein smearing can complicate comparison with endogenous bands.

How should I handle, dilute, and store recombinant CPA1 to preserve enzymatic activity?

The protein is shipped and stored at -20°C in single-use aliquots in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl₂. Do not refreeze after thawing — freeze-thaw cycling degrades activity. For working dilutions, use the storage buffer supplemented with 0.01% BSA to prevent adsorption to tube walls at low concentrations (<50 nM). Diluted enzyme on ice is stable for 4–6 hours; do not pre-dilute the day before. Unopened aliquots stored correctly are stable for 12 months from the date of receipt. Confirm activity with a Hip-Phe assay if the product has been stored for an extended period.