Carboxypeptidase A-6 (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant CPA6 on SDS-PAGE and Western blot?

The full-length human CPA6 protein is 437 amino acids, giving a predicted unmodified MW of ~49 kDa. Because this recombinant is expressed in HEK293 cells, N-linked glycosylation is intact, and the apparent MW on reducing SDS-PAGE typically runs at 55–62 kDa — consistent with published reports for the secreted, processed form. Under non-reducing conditions the band position may shift slightly. If you are running a Western blot positive control alongside RP-CarboxypeptidaseA6, load the 55–62 kDa window as your reference band.

Which processed isoform of CPA6 does this recombinant correspond to — pro-enzyme or mature form?

CPA6 is secreted as a zymogen with an N-terminal prodomain that is removed during activation. This recombinant is produced in HEK293 cells and supplied as the active, processed form lacking the inhibitory prodomain — analogous to the mature extracellular species detected in tissue and CSF studies. The signal peptide (residues 1–15, approximate) is also absent. If your experiment requires the zymogen for activation studies, note that this product will not serve that purpose; it is intended for direct enzymatic assays and as a calibrated positive control antigen.

What substrates work best for a fluorometric activity assay with recombinant Carboxypeptidase A-6?

CPA6 strongly prefers large hydrophobic C-terminal residues. The synthetic fluorogenic substrate N-(4-methoxyphenylazoformyl)-Phe-OH (MAPF) is well-established for M14-family carboxypeptidases and gives a clean linear signal with CPA6 in the nanomolar enzyme range. Hippuryl-Phe (Hippuryl-L-phenylalanine) is a reliable colorimetric alternative, read at 254 nm after TCA precipitation. Avoid substrates terminating in histidine, glycine, or alanine — CPA6 shows very weak activity toward these residues and signal-to-noise will be poor. Run a substrate-only blank in parallel to correct for any non-enzymatic hydrolysis.

What buffer conditions are optimal for CPA6 activity assays, and does the storage buffer interfere?

CPA6 activity assays are typically run in 25–50 mM HEPES or Tris-HCl, pH 7.5, supplemented with 100–150 mM NaCl and 5 µM ZnCl₂. The storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl₂) is compatible with these conditions; dilute the enzyme at least 1:10 into assay buffer to bring glycerol below 1%, which can otherwise reduce substrate diffusion and depress apparent kcat. CaCl₂ at low residual concentrations (≤0.5 mM after dilution) is well tolerated. Include 0.05% BSA if working below 5 nM enzyme to limit adsorptive losses.

What starting enzyme concentration should I use for a CPA6 kinetic assay?

For an initial Michaelis-Menten kinetic run with MAPF substrate, 2–10 nM recombinant CPA6 in a 100 µL reaction volume is a practical starting range. At this concentration, substrate depletion over a 30-minute read is less than 10% when [S] is set to 50–500 µM, keeping you within the linear portion of the velocity curve. If you are screening inhibitors for IC50 determination, fix enzyme at the lowest concentration that gives adequate signal (typically 2–5 nM) and verify that percent inhibition at your DMSO vehicle control is ≤0.5% at final DMSO ≤1%.

Can recombinant CPA6 be used as a positive control antigen for Western blot with the matched RP-CarboxypeptidaseA6 antibody?

Yes — this is a primary validated use case. RP-CarboxypeptidaseA6 (/anti-carboxypeptidase-a6-rabbit-polyclonal-antibody) was raised against the same CPA6 protein and is guaranteed compatible for Western blot detection. Load 20–50 ng of recombinant CPA6 per lane on a 10–12% SDS-PAGE gel; the antibody reliably detects the 55–62 kDa glycosylated band at primary dilutions of 1:500–1:2,000 in 5% non-fat milk/TBST. This pairing is particularly useful for antibody lot-to-lot validation and for confirming CPA6 identity in complex tissue lysate experiments alongside endogenous bands.

How much recombinant CPA6 should I load for a Western blot positive control lane?

20–50 ng is the recommended load range when using RP-CarboxypeptidaseA6 as the detecting antibody. At 50 ng you will see a strong, unambiguous band at 55–62 kDa suitable for size-marker alignment. At 20 ng the signal remains well within the antibody's linear detection range, which is preferable when you want the positive control band to be visually comparable to endogenous CPA6 in brain or kidney tissue lysates (typically low-to-moderate expressors). Dilute the recombinant into 1× Laemmli buffer immediately before use; do not store the diluted sample, as repeated boiling degrades activity and can affect band sharpness.

What is the shelf life of recombinant CPA6, and how should I handle it after thawing?

Stored at -20°C in the supplied single-use aliquots (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 5 mM CaCl₂), activity is stable for at least 12 months from the date of manufacture when freeze-thaw cycles are avoided. After thawing, keep the protein on ice and use within 4–6 hours; zinc-dependent metallocarboxypeptidases are sensitive to chelators and metal contamination introduced from repeated pipetting. Do not re-freeze a thawed aliquot. If you need a working stock for repeated assays within a week, dilute to 0.5–1 µM in assay buffer supplemented with 0.1% BSA and store at 4°C for no more than 5 days.