Carboxypeptidase D (Recombinant)

Frequently Asked Questions

What molecular weight does recombinant Carboxypeptidase D run at on SDS-PAGE?

The full-length human CPD protein is 1,380 amino acids, giving a predicted unmodified MW of ~154 kDa. However, because this recombinant is produced in HEK293 cells, extensive N-linked glycosylation adds significant mass — expect the apparent MW on reducing SDS-PAGE to run at approximately 180–200 kDa, consistent with the native gp180 designation. Anomalous migration relative to the sequence MW is normal and expected; use this recombinant as your molecular-weight reference when interpreting Western blot results with our matched antibody RP-CarboxypeptidaseD.

What processing forms or isoforms of Carboxypeptidase D should I expect in the recombinant preparation?

Native CPD undergoes limited proteolytic shedding and can exist as both the full-length transmembrane form and a soluble ectodomain fragment released from the cell surface. This HEK293-expressed recombinant is produced as the soluble ectodomain — encompassing the three extracellular metallocarboxypeptidase-like domains — rather than the full-length type I transmembrane protein. It lacks the transmembrane anchor and cytoplasmic tail. Researchers studying membrane trafficking through the trans-Golgi or endosomal compartments should bear this distinction in mind when designing topology-sensitive assays.

What substrates does recombinant Carboxypeptidase D cleave and what is a good activity assay substrate?

CPD (EC 3.4.17.22) is a metallocarboxypeptidase that removes C-terminal basic residues — arginine and lysine — from peptides and proteins. For fluorometric activity assays, hippuryl-Arg (Hip-Arg) and hippuryl-Lys (Hip-Lys) are well-established colorimetric/fluorescent substrates; Hip-Arg is generally preferred because CPD shows higher efficiency toward C-terminal arginine. Dansyl-Ala-Arg or Z-Ala-Arg can also be used in HPLC-based release assays. Monitor activity by measuring hippuric acid release at 254 nm or using a coupled fluorescence format. Include 5 mM CaCl2 in the assay buffer to support metalloprotease activity.

What buffer conditions are optimal for Carboxypeptidase D activity assays?

The recombinant is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, and 5 mM CaCl2. For activity assays, this buffer is directly compatible — dilute the enzyme into the same or similar buffer at pH 7.4–7.5. CaCl2 is essential; do not use EDTA or EGTA-containing buffers, as chelation of the catalytic zinc and accessory calcium ions will abolish activity. Reducing agents such as DTT at low concentrations (≤1 mM) are generally tolerated. Substrate solubility permitting, keep assay volume ≥50 µL to ensure reliable pipetting of dilute enzyme stocks.

What starting concentration of recombinant CPD should I use for an enzyme activity assay?

As a practical starting point, titrate the recombinant CPD in the range of 0.1–1 µg per reaction (50–100 µL total volume) using Hip-Arg substrate at 1–2 mM. Activity is linear with enzyme concentration over roughly one order of magnitude under these conditions. If you are measuring inhibitor IC50 values, fix enzyme concentration at the lower end of the linear range (~0.2 µg/reaction) to avoid substrate depletion artifacts. Always run a no-enzyme blank to account for non-enzymatic substrate hydrolysis, particularly with hippuryl substrates at longer incubation times (>30 min).

Can I use recombinant Carboxypeptidase D as a positive control for Western blot with the matched antibody?

Yes — this is a primary intended use case. The matched rabbit polyclonal antibody RP-CarboxypeptidaseD (/anti-carboxypeptidase-d-rabbit-polyclonal-antibody) was validated against this recombinant and is guaranteed compatible for Western blot. Load 50–100 ng of recombinant CPD per lane under reducing conditions; expect a band at ~180–200 kDa reflecting the glycosylated ectodomain. This provides a clean, unambiguous positive control band when probing cell or tissue lysates where endogenous CPD signal may be variable. The antibody has been validated for Western blot in TPB's laboratory.

How much recombinant Carboxypeptidase D should I load as a positive control for antibody validation experiments?

For antibody validation alongside the matched RP-CarboxypeptidaseD antibody, 50 ng per lane is sufficient to produce a robust signal on standard ECL-based Western blots with HRP-conjugated secondary antibodies at typical dilutions. If you are titrating primary antibody concentration or comparing lot-to-lot antibody performance, prepare a 3-point dilution series of the recombinant (25, 50, 100 ng/lane) to establish linearity of detection. This recombinant-antibody pairing is particularly useful for IHC antigen-retrieval optimization, where confirmed recombinant protein spotted on a nitrocellulose strip serves as a reference standard.

How should I store and handle recombinant Carboxypeptidase D to preserve enzymatic activity?

Upon receipt, store at -20°C in single-use aliquots immediately — do not store at 4°C long-term, as metallocarboxypeptidases are susceptible to autolytic degradation and activity loss over days at refrigerator temperature. Avoid repeated freeze-thaw cycles; each cycle measurably reduces specific activity. For working dilutions, prepare fresh in assay buffer immediately before use and keep on ice. The supplied buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl2) stabilizes the enzyme during storage. Purity is >90% by SDS-PAGE and endotoxin is <0.1 EU/µg by LAL test.