Carboxypeptidase B-1 (Recombinant)

Frequently Asked Questions

What molecular weight does recombinant Carboxypeptidase B-1 run at on SDS-PAGE?

The mature, active form of human CPB1 (UniProt P15086) spans roughly residues 96–417 after propeptide removal, giving a predicted MW of ~35 kDa. On reducing SDS-PAGE, this recombinant produced in HEK293 cells typically migrates at 37–40 kDa — the slight upward shift from the theoretical value reflects N-linked glycosylation contributed by the mammalian expression system. If you are using this as a Western blot size reference, expect the band at approximately 38 kDa under standard reducing conditions.

Is the recombinant CPB1 supplied as the zymogen or the active mature enzyme?

It is supplied as the active mature enzyme, not the inactive procarboxypeptidase B zymogen. The N-terminal propeptide (approximately residues 1–95 of the 417 aa precursor) has been removed during processing, yielding the catalytically competent form. This matters if you are running activity assays or using the protein as an enzymatically active standard — no additional activation step is required. If your assay specifically requires the proenzyme form, this product is not the appropriate choice.

What substrates does Carboxypeptidase B-1 cleave and which should I use for an activity assay?

CPB1 (EC 3.4.17.2) is a zinc-dependent metallocarboxypeptidase that selectively hydrolyzes basic C-terminal residues — arginine and lysine — from peptide substrates. For in vitro activity assays, the chromogenic substrate hippuryl-L-arginine (Hip-Arg) is the most widely used, monitored by absorbance at 254 nm following hippuric acid release. Fluorogenic alternatives such as Abz-Ala-Arg-OH offer better sensitivity in low-volume formats. CPB1 shows negligible activity toward C-terminal aromatic or aliphatic residues, making substrate selectivity a useful quality check when validating lot activity.

What buffer conditions are optimal for CPB1 activity assays with this recombinant?

CPB1 is a zinc-dependent metallocarboxypeptidase, so the assay buffer must preserve the active-site zinc. The protein is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol with 5 mM CaCl2. For activity assays, dilute into a compatible reaction buffer — 50 mM Tris-HCl pH 7.5 or 25 mM HEPES pH 7.4, 100–150 mM NaCl, 5 mM CaCl2 — and keep pH between 7.2 and 8.0. Avoid EDTA or EGTA; even low micromolar concentrations will chelate the catalytic zinc and abolish activity. DTT at ≤1 mM is generally tolerated.

What starting concentration of recombinant CPB1 should I use for a peptidase activity assay?

A reasonable starting point for a Hip-Arg chromogenic assay is 1–5 nM enzyme in a 100–200 µL reaction volume, with substrate in the 0.1–1 mM range to bracket Km. Adjust enzyme concentration to achieve linear initial-rate conditions — typically 5–10% substrate conversion over the measurement window. For fluorogenic Abz-Ala-Arg assays, 0.5–2 nM is usually sufficient given the higher sensitivity. Always run a no-enzyme blank to account for spontaneous substrate hydrolysis, and equilibrate the reaction mix to 37°C before adding enzyme.

Can I use recombinant CPB1 as a positive control for Western blot with the matched RP-CarboxypeptidaseB1 antibody?

Yes — this is one of the primary use cases for pairing REC-CarboxypeptidaseB1 with RP-CarboxypeptidaseB1. The rabbit polyclonal antibody was raised and validated in the same laboratory against CPB1 protein, ensuring the recombinant is recognized reliably on Western blot. Load 10–50 ng of recombinant CPB1 per lane alongside your cell or tissue lysate. The antibody is validated for Western blot, so you should see a clean band at ~38 kDa. This pairing also serves as an antibody validation positive control when assessing new lots of RP-CarboxypeptidaseB1.

How much recombinant Carboxypeptidase B-1 should I load as a Western blot positive control?

Load 20–50 ng per lane as a starting point when using the matched RP-CarboxypeptidaseB1 antibody (SKU: RP-CarboxypeptidaseB1). At this range, the ~38 kDa band is consistently detectable without saturating the signal or obscuring nearby bands in adjacent lysate lanes. If your secondary antibody or detection system is high-sensitivity (e.g., HRP with femtomolar chemiluminescence substrates), titrate down to 5–10 ng. Dilute the recombinant in 1× SDS loading buffer immediately before use; avoid repeated dilution from the same aliquot to minimize activity and protein loss.

How should I store and handle recombinant CPB1 to maintain activity over time?

Store at −20°C in single-use aliquots. The supplied buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl2 — is formulated for freeze-thaw stability, but repeated freeze-thaw cycles will progressively degrade metalloprotease activity due to zinc cofactor dissociation and protein aggregation. Upon first use, thaw one aliquot on ice, take what you need, and discard the remainder rather than refreezing. For short-term working stocks (1–3 days), keep at 4°C. Endotoxin is <0.1 EU/µg by LAL assay, making it suitable for cell-based applications where LPS contamination is a concern.