PCOLE-1 (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of PCOLE-1 (Recombinant) on SDS-PAGE and Western blot?

The full-length PCOLCE-encoded protein is 449 amino acids (predicted unglycosylated MW ~50 kDa), but because this recombinant is expressed in HEK293 cells under native-like glycosylation conditions, the observed band on reducing SDS-PAGE typically migrates at approximately 55–65 kDa. The shift reflects N-linked glycosylation characteristic of the secreted, mature ECM form. Expect a diffuse or slightly smeared band rather than a sharp single band — this is normal for glycoproteins and is not indicative of degradation. Deglycosylation with PNGase F collapses the band to ~50 kDa.

Is PCOLE-1 (Recombinant) the full-length protein or the mature secreted form lacking the signal peptide?

This recombinant corresponds to the mature secreted form of human PCPE-1 (UniProt Q15113). The N-terminal signal peptide is cleaved co-translationally during HEK293 expression, so what is recovered from conditioned medium is the processed extracellular form — the biologically relevant species for collagen maturation studies. No additional prodomain processing is required. The two CUB domains and the NTR domain are intact, preserving the conformation-sensitive binding interface with the C-terminal propeptide of fibrillar procollagens.

Does PCOLE-1 (Recombinant) have enzymatic activity itself, or does it require BMP-1/tolloid for collagen C-propeptide cleavage?

PCPE-1 is a non-catalytic enhancer — it has no intrinsic proteinase activity. Its function is to bind the C-terminal propeptide of fibrillar procollagens (best characterized for type I procollagen) and accelerate cleavage by BMP-1/tolloid family metalloproteinases. In activity assays, PCOLE-1 (Recombinant) should be used alongside a source of active BMP-1 or mTLD. Without the metalloproteinase, you will not observe substrate processing regardless of PCPE-1 concentration. This recombinant is validated as an enhancer, not a standalone proteinase.

What activity assay can I use to measure PCOLE-1 (Recombinant) enhancement of procollagen C-proteinase, and what substrate should I use?

The standard assay uses recombinant type I procollagen C-propeptide (pCP) or mini-procollagen substrates incubated with a titrated amount of active BMP-1 in the presence or absence of PCOLE-1 (Recombinant). Cleavage is monitored by SDS-PAGE densitometry or fluorescent-tagged substrate variants. A typical starting point is 50–200 ng/mL PCOLE-1 alongside 10–50 ng/mL BMP-1 in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM CaCl₂ at 37°C for 1–4 hours. Enhancement is expressed as fold-increase in cleavage rate relative to BMP-1 alone. Titrate PCPE-1 to establish dose-dependence before committing to a fixed concentration.

What buffer conditions are optimal for PCOLE-1 (Recombinant) activity assays and protein–protein interaction studies?

The storage buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol — is compatible with most collagen maturation assays and can be used directly. For SPR or pull-down binding assays measuring PCPE-1 interaction with procollagen C-propeptide, reduce glycerol to ≤1% by diluting into assay buffer immediately before use. CaCl₂ at 1–5 mM is recommended when BMP-1 is a binding partner, as it is required for metalloproteinase activity. Avoid EDTA or EGTA in functional assays. pH should remain between 7.2 and 7.8; PCPE-1 binding interactions are conformation-sensitive and activity drops sharply below pH 6.8.

What starting concentration of PCOLE-1 (Recombinant) is recommended for co-immunoprecipitation or pull-down with BMP-1?

For co-IP or GST/His-tag pull-down experiments, a practical starting range is 0.5–2 µg PCOLE-1 (Recombinant) per reaction. Because PCPE-1 binds the procollagen C-propeptide non-covalently with moderate affinity, keeping protein concentrations in the 100–500 nM range during incubation improves complex recovery. Include 0.05% Tween-20 and 1 mg/mL BSA in binding buffer to reduce non-specific adsorption. Verify the pulled-down PCPE-1 by Western blot using our matched antibody RP-PCOLE1, which is validated for WB and provides a reliable readout at these loading amounts.

Can I use PCOLE-1 (Recombinant) as a positive control for Western blot with the matched antibody RP-PCOLE1?

Yes — PCOLE-1 (Recombinant) and antibody RP-PCOLE1 are produced from the same lab and are guaranteed compatible for Western blot positive control applications. Load 25–100 ng of recombinant protein per lane on a 10–12% SDS-PAGE gel under reducing conditions. The antibody at 1:1,000–1:2,000 dilution (primary, overnight 4°C) detects a band at ~55–65 kDa, consistent with the glycosylated mature form. This serves as a reliable size marker and signal calibrator when running cell lysates or tissue extracts alongside. Product page and protocol for RP-PCOLE1 are at /anti-pcole-1-rabbit-polyclonal-antibody.

How should I handle, dilute, and store PCOLE-1 (Recombinant) to maintain activity over multiple experiments?

PCOLE-1 (Recombinant) is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol as single-use aliquots. Store at -20°C; purity is >95% by SDS-PAGE and endotoxin is <0.1 EU/µg by LAL. Avoid repeated freeze-thaw — each cycle risks glycoprotein aggregation and loss of the native conformation required for procollagen C-propeptide binding. For working dilutions, prepare fresh in assay buffer the day of the experiment and keep on ice. If you anticipate multiple uses within a week, a single thaw followed by storage at 4°C for up to 5 days is acceptable; beyond that, re-aliquot before first thaw.