Carboxypeptidase A-4 (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant Carboxypeptidase A-4 on SDS-PAGE?

The full-length human CPA4 protein (UniProt Q9UI42) encodes 421 amino acids, giving a calculated molecular weight of approximately 47 kDa. Because this recombinant is produced in HEK293 mammalian cells, N-linked glycosylation is expected, and the apparent band on reducing SDS-PAGE typically migrates at 50–55 kDa. If you are running a Western blot positive control alongside our matched antibody (RP-CarboxypeptidaseA4), expect the dominant band in that 50–55 kDa window. A faint pro-form band may appear slightly higher depending on processing efficiency in the HEK293 expression system.

Is recombinant CPA4 expressed as a zymogen or an active mature form?

CPA4, like other secreted A-type carboxypeptidases, is synthesized as an inactive proenzyme carrying an N-terminal propeptide that occludes the active site. This recombinant is produced in HEK293 cells under conditions that promote secretion and propeptide processing, yielding predominantly the mature, active form. The mature chain runs slightly smaller than the full-length 421-aa sequence on SDS-PAGE (~47 kDa calculated, ~50–55 kDa apparent with glycosylation). If your experimental model requires the zymogen form specifically, contact us — but for standard activity assays, this product is supplied ready to use as an active enzyme.

What substrates does recombinant Carboxypeptidase A-4 cleave in a fluorometric activity assay?

CPA4 preferentially cleaves hydrophobic C-terminal residues, with documented selectivity for -Phe, -Leu, -Ile, -Met, -Tyr, and -Val. For fluorometric assays, the synthetic substrate N-(4-methoxyphenylazoformyl)-Phe-OH (MAPF) is widely used and compatible with CPA4's substrate preference. Abz-Gly-Phe(NO2) peptide substrates are also reported. Run initial Km determinations at substrate concentrations ranging from 10 µM to 1 mM in assay buffer. Fluorescence should be monitored at excitation ~320 nm / emission ~420 nm depending on your fluorophore pair. Include 1 mM EDTA as a no-zinc negative control to confirm zinc-dependent activity.

What activity assay buffer should I use for recombinant CPA4 and does the storage buffer interfere?

A standard assay buffer for CPA4 activity is 50 mM Tris-HCl pH 7.5, 100–150 mM NaCl, 0.5–1 mM ZnCl2. The product storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl2) is generally compatible at dilutions ≥1:10 into assay buffer; glycerol carry-over below 1% (v/v) does not meaningfully inhibit activity. Avoid EDTA or EGTA in assay buffers, as chelation of the catalytic zinc ion abolishes activity. If you need to switch buffers, dialysis or a desalting spin column equilibrated in assay buffer is appropriate prior to Km/kcat determination.

What starting enzyme concentration should I use for a CPA4 inhibitor IC50 assay?

For IC50 determinations, we recommend titrating the enzyme to identify the concentration that produces a linear signal increase over 30–60 minutes under your substrate conditions. A practical starting point is 0.5–5 nM recombinant CPA4 with substrate at or below the Km (typically 50–200 µM for MAPF-type substrates). Using enzyme well below substrate concentration avoids substrate depletion artifacts and keeps you in the linear regime required for accurate Ki extrapolation. Pre-incubate inhibitor with enzyme for 15–30 minutes at 37°C before substrate addition to capture slow-binding inhibitors. Run at minimum in triplicate with a DMSO vehicle control ≤0.5% (v/v).

Can I use recombinant Carboxypeptidase A-4 as a Western blot positive control for the RP-CarboxypeptidaseA4 antibody?

Yes — this is one of the primary intended uses of REC-CarboxypeptidaseA4 alongside RP-CarboxypeptidaseA4 (/anti-carboxypeptidase-a4-rabbit-polyclonal-antibody). The matched rabbit polyclonal antibody was raised and validated in the same lab, and the two products are guaranteed compatible for Western blot positive control experiments. Load 10–50 ng of recombinant CPA4 per lane on a standard 10–12% SDS-PAGE gel. Under reducing conditions, expect a band at 50–55 kDa. This provides a clean, reproducible positive control lane that confirms antibody performance and helps calibrate signal intensity against your cell lysate or tissue samples.

How much recombinant CPA4 should I load per lane for Western blot, and at what antibody dilution?

Start with 20–50 ng of REC-CarboxypeptidaseA4 per lane. Because HEK293-expressed recombinant is a defined, purified protein (>90% by SDS-PAGE), it produces a strong, clean band compared to endogenous CPA4 in tissue lysates, so titrate down if signal is saturating. Pair with RP-CarboxypeptidaseA4 at the dilution specified on that product's datasheet (typically 1:500–1:2000 in 5% non-fat milk or BSA/TBST). Run the recombinant control in an adjacent lane to your experimental samples. Use HRP-conjugated anti-rabbit secondary at 1:5,000–1:10,000. Confirm that the band co-migrates with your endogenous CPA4 signal.

How should I store and handle recombinant CPA4 to preserve enzymatic activity long-term?

REC-CarboxypeptidaseA4 is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl2, and shipped on dry ice. Upon receipt, store at -20°C in the single-use aliquots provided. Avoid repeated freeze-thaw cycles, as they progressively reduce metalloexopeptidase activity — each cycle can cause measurable losses. For experiments requiring multiple uses from the same preparation, prepare working dilutions in assay buffer, keep on ice, and discard unused diluted material at end of session. Do not add carrier BSA unless specifically needed, as it can interfere with downstream activity or binding assays. Shelf life is 12 months from receipt when stored correctly.