PC-6 (Recombinant)

Frequently Asked Questions

What molecular weight band should I expect for recombinant PC-6 on SDS-PAGE or Western blot?

REC-PC6 is produced in HEK293 cells and undergoes autocatalytic zymogen processing and N-linked glycosylation, so the mature, active form migrates at approximately 85–95 kDa under reducing SDS-PAGE conditions — noticeably higher than the ~75 kDa predicted from the processed polypeptide sequence alone. Glycosylation accounts for the difference and can produce a diffuse or doublet appearance. If you observe a band near 110 kDa, this likely reflects incompletely processed or heterogeneously glycosylated species, which are normal minor components at >90% purity by SDS-PAGE.

What processing state is REC-PC6 — is it the full-length zymogen or the mature active convertase?

REC-PC6 is secreted by HEK293 cells as the autocatalytically processed, mature active form. During transit through the constitutive secretory pathway, the propeptide is cleaved intramolecularly at its paired basic site, and the resulting non-covalent propeptide–catalytic domain complex dissociates under the mildly acidic conditions of the trans-Golgi network. The purified product therefore represents the enzymatically competent catalytic domain, not the full 969-amino-acid zymogen. If your assay requires the zymogen form as a control, this product is not the appropriate reagent.

What substrates and cleavage motifs does recombinant PC-6 recognize in an in vitro activity assay?

PC-6 cleaves after the C-terminal basic residue of RXXX[KR]R consensus motifs. For fluorogenic activity assays, the synthetic peptide substrate Pyr-RTKR-AMC (also used for furin and other PCSK family members) is a well-characterized choice; PC-6 hydrolyzes it with a Km in the low-micromolar range. PCSK6-specific proproteins such as BMP-4, PDGF-A, and VEGF-C precursors can serve as physiological substrates in gel-based cleavage assays. Always include a furin-specific inhibitor control (e.g., dec-RVKR-CMK) to confirm PCSK family-dependent activity in your system.

What buffer conditions are optimal for a PC-6 activity assay, and what starting enzyme concentration should I use?

REC-PC6 is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol and is active under these conditions. For fluorogenic peptide assays, a reaction buffer of 100 mM HEPES pH 7.0–7.5, 1 mM CaCl₂, and 0.1% Tween-20 at 37 °C is widely reported and compatible. A starting enzyme concentration of 1–5 nM is reasonable for initial Km determinations with Pyr-RTKR-AMC; titrate substrate from 10 µM to 500 µM. Avoid chelators such as EDTA, as calcium supports the subtilisin-like fold stability of PCSK family members.

How should I calculate IC50 values for PC-6 inhibitors using this recombinant, and are there known artifacts to watch for?

Establish a linear velocity window first — confirm enzyme concentration and substrate concentration produce a linear fluorescence increase for at least 30 minutes before introducing inhibitor. Use substrate concentrations at or near Km to avoid substrate competition artifacts that inflate apparent IC50 values. Because many published PCSK inhibitors (including peptide-based CMK derivatives) are irreversible, report ki,inact rather than IC50 for covalent compounds. Keep glycerol ≤5% in the final assay volume, as the storage buffer contains 10% glycerol that can mildly affect peptide substrate solubility at higher enzyme input volumes.

Can I use REC-PC6 as a positive control for Western blot with the matched RP-PC6 antibody?

Yes — REC-PC6 and the matched rabbit polyclonal antibody RP-PC6 (see /anti-pc-6-rabbit-polyclonal-antibody) are produced and validated in the same laboratory, making them a reliable pairing. Load 20–50 ng of REC-PC6 per lane alongside your cell or tissue lysate. The antibody is validated for Western blot and should detect the ~85–95 kDa mature band under standard reducing conditions. This is particularly useful when confirming endogenous PC-6 expression in non-neuroendocrine cell lysates, where signal can be weak and a defined-mass positive control is essential for band assignment.

How much recombinant PC-6 should I load to serve as a positive control when validating RP-PC6 antibody by Western blot?

For a clean, unambiguous positive control lane, 25–50 ng of REC-PC6 per lane is typically sufficient with RP-PC6 at a 1:1,000–1:2,000 dilution and standard ECL detection. Starting at 50 ng is recommended during initial antibody titer optimization; once the linear detection range is established, 20–25 ng is usually adequate. Dilute REC-PC6 directly into 1× Laemmli sample buffer containing β-mercaptoethanol, heat at 95 °C for 5 minutes, and load immediately. Avoid loading >100 ng per lane, as overloading can obscure nearby endogenous bands in co-loaded lysate lanes.

What is the shelf life of REC-PC6, and how should I handle it after first thaw to preserve enzymatic activity?

REC-PC6 is stable for 12 months from the date of receipt when stored at -20 °C in the supplied single-use aliquots. After thawing, keep the protein on ice and use within the same working session; enzymatic activity decreases measurably with repeated freeze-thaw cycles. If sub-aliquoting is necessary, add carrier protein (0.1% BSA, protease-free) to prevent surface adsorption losses at low concentrations (<10 µg/mL). Do not store working dilutions at 4 °C for more than 24 hours. Endotoxin is <0.1 EU/µg by LAL, so no additional endotoxin removal is required for standard biochemical assays.