Carboxypeptidase B-2 (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant human CPB2/TAFI on SDS-PAGE?

The full-length CPB2 precursor (423 aa, UniProt Q96IY4) has a predicted polypeptide MW of ~48 kDa, but N-linked glycosylation in HEK293 cells shifts the apparent band to ~55–60 kDa under reducing SDS-PAGE conditions. This is consistent with the native plasma zymogen. If you activate with thrombin–thrombomodulin, the activation peptide (residues 1–92) is cleaved, and TAFIa migrates at ~35–38 kDa. Always run both forms if your experiment requires distinguishing zymogen from active enzyme.

Is this recombinant CPB2 supplied as zymogen or already activated TAFIa?

The protein is supplied as the intact zymogen form, equivalent to the circulating plasma precursor. For activity assays, you must activate it in vitro. Thrombin–thrombomodulin (typically 10 nM thrombin + 50 nM soluble thrombomodulin) is the physiologically relevant activator and is recommended for mechanistic studies. Trypsin (10:1 substrate:trypsin molar ratio, 37°C, 10–15 min) is a faster alternative for generating active enzyme in screening workflows. Confirm activation by loss of the ~55–60 kDa band and appearance of ~35–38 kDa TAFIa on SDS-PAGE.

Which synthetic substrates work best for measuring recombinant TAFIa carboxypeptidase activity?

Hippuryl-L-arginine (Hippuryl-Arg) and Ala-Ala-Phe-Arg (AAFR) are the most widely validated chromogenic substrates for TAFIa (EC 3.4.17.20). Hippuryl-Arg is cleaved to yield hippuric acid, measurable at 254 nm. Typical assay conditions: 0.5–2 nM activated TAFIa, 1–5 mM Hippuryl-Arg, 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 37°C. The recombinant's HEK293-derived glycosylation is preserved, so kinetic parameters (Km ~1–2 mM for Hippuryl-Arg) align with reported values for plasma-derived TAFI.

What buffer conditions should I use for TAFIa inhibitor IC50 screening with this recombinant?

Run IC50 assays in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, which matches the supplied storage buffer and minimizes dilution artifacts. Keep activated TAFIa at 0.5–1 nM (well below Km) to satisfy Morrison tight-binding criteria if testing potent inhibitors. Add 0.01% Tween-20 or 0.1% BSA to suppress non-specific adsorption at low enzyme concentrations. TAFIa is thermolabile (half-life ~8–15 min at 37°C, pH 7.4); prepare enzyme immediately before addition and maintain consistent incubation times across replicates to reduce assay variability.

How much recombinant Carboxypeptidase B-2 should I load as a Western blot positive control?

For a positive control lane using the matched antibody RP-CarboxypeptidaseB2, load 20–50 ng of recombinant CPB2 zymogen per lane. This consistently produces a clean band at ~55–60 kDa under reducing conditions with standard ECL detection. If your sample matrix contains abundant plasma proteins, load 50 ng to ensure the recombinant band is unambiguous. Pair with RP-CarboxypeptidaseB2 (see /anti-carboxypeptidaseb2-rabbit-polyclonal-antibody) at 1:1,000–1:2,000 dilution; this combination was validated in-house and is the most reliable way to confirm antibody lot-to-lot consistency.

Can I use recombinant CPB2 to validate RP-CarboxypeptidaseB2 antibody specificity by Western blot?

Yes, and this is the intended use case. RP-CarboxypeptidaseB2 is a rabbit polyclonal raised against human CPB2 and is validated for Western blot by the same team that produced this recombinant. Loading a titration of 10, 25, and 50 ng of REC-CarboxypeptidaseB2 alongside your experimental lysates confirms antibody sensitivity and linear range in your specific detection system. A single band at ~55–60 kDa in the recombinant lane, with no spurious bands, constitutes a clean specificity control. Details and pairing data are on the antibody page at /anti-carboxypeptidaseb2-rabbit-polyclonal-antibody.

How should I handle and store recombinant CPB2/TAFI to preserve zymogen integrity?

Upon receipt, aliquot into single-use volumes at the intended working concentration and store at −20°C. The supplied buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol) is formulated to protect against freeze-thaw denaturation, but repeated cycling still accelerates spontaneous activation and aggregation. Do not store working aliquots at 4°C for more than 48 hours, as the zymogen undergoes gradual autolytic activation at this temperature. Avoid diluting below 10 µg/mL without adding carrier protein (0.1% BSA) to prevent surface adsorption losses. Purity is >90% by SDS-PAGE; endotoxin is <0.1 EU/µg by LAL.

Does HEK293 expression affect CPB2 glycosylation compared to native plasma TAFI?

HEK293 cells produce complex N-linked glycans on CPB2 at the same three occupied sequons reported for native plasma TAFI (N219, N356, and N369), making the glycosylation pattern substantially more native-like than insect- or E. coli-derived material. This matters for thrombin–thrombomodulin activation studies, where the activation peptide's glycan at N219 influences activation kinetics. However, minor differences in sialylation between HEK293 and hepatocyte-derived protein are possible. If exact glycan structure is critical to your experiment, confirm with lectin blotting. For most activation kinetics, inhibitor screening, and immunoassay calibration applications, HEK293 expression is appropriate.