Carboxypeptidase Z (Recombinant)

Frequently Asked Questions

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

The full-length human CPZ protein is 652 amino acids, giving a predicted unmodified MW of ~73 kDa. Because this recombinant is produced in HEK293 cells, N-linked glycosylation adds measurable mass: expect a diffuse band running at approximately 80–90 kDa under reducing SDS-PAGE conditions, consistent with the glycosylation pattern of the native secreted protein. Under non-reducing conditions, intramolecular disulfide bonds in the cystatin-like domain may shift migration slightly. When blotting with RP-CarboxypeptidaseZ, target the 80–90 kDa window for detection.

What processing form of Carboxypeptidase Z does the recombinant represent — pro-form or mature enzyme?

The recombinant material represents the secreted, processed form of CPZ rather than an intracellular zymogen. CPZ contains an N-terminal signal peptide and a prodomain that are cleaved during secretion; HEK293 expression recapitulates this processing, yielding predominantly the mature enzyme competent for zinc coordination and substrate binding. If your experiment requires the latent pro-form, additional precautions or alternative constructs would be needed. For substrate cleavage assays and most binding studies, the HEK293-derived material can be used directly without further activation steps.

What substrates does Carboxypeptidase Z cleave and which are suitable for a fluorogenic activity assay?

CPZ is a zinc-dependent metallocarboxypeptidase (EC 3.4.17.-) that preferentially cleaves C-terminal basic residues, with a strong preference for arginine over lysine. For fluorogenic activity assays, dansyl-Ala-Arg is a well-established small-molecule substrate for the M14 carboxypeptidase family and provides a convenient readout at Ex/Em ~340/520 nm. Hippuryl-Arg (hippuryl-L-arginine) is a chromogenic alternative monitored at 254 nm. Start with substrate concentrations in the 0.1–1 mM range and titrate enzyme from 1–10 nM to establish linear kinetics before running inhibitor or mutant comparisons.

What buffer conditions are recommended for Carboxypeptidase Z activity assays with this recombinant?

The recombinant is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, and 5 mM CaCl₂ — the CaCl₂ is included because CPZ is a metalloproteinase and divalent cations support structural stability. For activity assays, a compatible reaction buffer is 50 mM HEPES pH 7.5, 100 mM NaCl, 5 mM CaCl₂. Avoid EDTA or EGTA, which chelate the catalytic zinc and will abolish activity. DTT at concentrations above 1 mM can also interfere with disulfide-dependent folding of the cystatin-like domain; keep reducing agents minimal or absent during assays.

What starting concentration of recombinant Carboxypeptidase Z should I use for a substrate cleavage assay?

A reasonable starting point is 2–5 nM enzyme in your reaction well when using a fluorogenic substrate such as dansyl-Ala-Arg at 200 µM. Under these conditions, a 60-minute room-temperature incubation typically yields a quantifiable signal with a good signal-to-background ratio. If you are screening inhibitors and need a robust assay window, titrate enzyme from 1 to 20 nM and choose the concentration that gives 20–40% substrate conversion in 30 minutes — this keeps the assay in the linear range and reduces substrate depletion artifacts that confound IC₅₀ determinations.

Can I use recombinant Carboxypeptidase Z as a positive control for Western blot with the matched TPB antibody RP-CarboxypeptidaseZ?

Yes — this is one of the primary intended uses. The recombinant (REC-CarboxypeptidaseZ) and the matched rabbit polyclonal antibody (RP-CarboxypeptidaseZ) are developed in the same lab and guaranteed compatible. Loading 10–50 ng of recombinant protein per lane alongside your cell lysate samples gives a clean, reproducible band at ~80–90 kDa under reducing conditions. This positive control is especially useful when validating RP-CarboxypeptidaseZ against a new cell line or tissue lysate, confirming antibody performance independently of endogenous expression levels.

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

Load 20–50 ng per lane as a starting point; this reliably produces a detectable band with RP-CarboxypeptidaseZ at a primary antibody dilution of 1:1,000–1:5,000. If your film or imager tends toward high background, drop to 10 ng and confirm linearity. Because the HEK293-expressed protein is glycosylated, the band will appear at ~80–90 kDa rather than the sequence-predicted ~73 kDa — account for this when comparing to molecular weight markers so you do not misidentify the band. Run the recombinant under the same reducing conditions as your experimental samples for a fair comparison.

How should I store and handle recombinant Carboxypeptidase Z to maintain activity over time?

The protein is supplied as a ready-to-use solution in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl₂ and should be stored at −20°C in single-use aliquots. Repeated freeze-thaw cycles progressively reduce metallocarboxypeptidase activity by disrupting zinc coordination; aliquot on receipt to avoid this. On the day of use, thaw on ice and briefly centrifuge to pellet any microaggregates. Working stocks diluted in assay buffer can be held on ice for up to 4 hours; do not leave the enzyme at 37°C for extended periods outside an active assay. Purity is >90% by SDS-PAGE and endotoxin is <0.1 EU/µg by LAL.