PC-9 (Recombinant)

Frequently Asked Questions

What molecular weight does recombinant PCSK9 run at on SDS-PAGE and Western blot?

Full-length human PCSK9 has a predicted MW of ~72 kDa, but the HEK293-expressed PC-9 (Recombinant) typically migrates at ~75–78 kDa under reducing SDS-PAGE conditions due to N-linked glycosylation. The propeptide (~14 kDa) remains non-covalently associated under native conditions but dissociates under denaturing conditions, so you may observe a secondary band near 14 kDa at higher loads. On Western blot with RP-PC9, expect the primary band at 75–78 kDa and confirm autocatalytic processing by the presence or absence of the 14 kDa propeptide band.

Is recombinant PCSK9 supplied as the full-length precursor or the mature autocatalytically processed form?

PC-9 (Recombinant) is produced in HEK293 cells and undergoes autocatalytic cleavage in the endoplasmic reticulum, mirroring the in vivo processing of endogenous PCSK9. The product is the propeptide-bound mature form — the biologically relevant circulating species — not the unprocessed precursor. The propeptide (residues 31–152) remains non-covalently associated with the catalytic domain. This is important for binding assays: the propeptide-occupied active site means the protein behaves as it does in serum, making it directly suitable for LDLR-interaction and inhibitor studies without additional processing steps.

What substrate or assay can I use to measure PCSK9 activity or LDLR binding in vitro?

PCSK9 does not function as a classical protease toward external substrates in the canonical sense; its primary activity is non-catalytic LDLR degradation mediated by direct EGF-A domain binding. For binding assays, surface plasmon resonance (SPR) or ELISA-based LDLR EGF-A domain competition is the standard approach. Use 50–500 nM PC-9 (Recombinant) in PBS or 50 mM Tris-HCl pH 7.4, 150 mM NaCl against immobilized or soluble LDLR EGF-A domain (residues 293–337). For cell-based degradation assays, 1–5 µg/mL added to LDLR-expressing hepatic cell lines (e.g., HepG2) is a well-established functional range.

What buffer conditions are recommended for PCSK9 LDLR-binding or inhibitor competition assays?

PC-9 (Recombinant) is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol. For LDLR EGF-A binding assays, dilute into assay buffer of PBS pH 7.4 or 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.05% Tween-20, 0.1% BSA — glycerol carry-over from storage buffer at ≤1% (v/v) final does not measurably affect binding affinity. PCSK9–LDLR interaction is pH-sensitive: binding is strong at pH 7.4 (mimicking plasma) and substantially weaker at endosomal pH ~5.4, so maintain pH control in competition assays when evaluating acidic-environment inhibitors.

How much recombinant PCSK9 should I load as a Western blot positive control with the RP-PC9 antibody?

For Western blot positive controls paired with RP-PC9 (/anti-pc-9-rabbit-polyclonal-antibody), load 20–50 ng of PC-9 (Recombinant) per lane under reducing conditions. At 50 ng, the primary band at 75–78 kDa is clearly resolved with standard ECL detection at RP-PC9 dilutions of 1:1,000–1:2,000. Loading less than 20 ng may require higher antibody concentrations or more sensitive substrates. Because both the recombinant protein and RP-PC9 are produced from the same target sequence in the same lab, this pairing provides a reliable, consistent positive control lane for each blot run.

Can I use PC-9 (Recombinant) to validate the RP-PC9 rabbit polyclonal antibody for Western blot or IHC?

Yes — this is one of the primary design intentions of the TPB matched-reagent system. PC-9 (Recombinant) and RP-PC9 (/anti-pc-9-rabbit-polyclonal-antibody) are developed against the same human PCSK9 sequence and validated together in-house. For Western blot antibody validation, 20–50 ng of recombinant loaded alongside cell lysate (e.g., HepG2 or HEK293 overexpressing PCSK9) confirms antibody specificity at the expected 75–78 kDa. For IHC validation workflows, the recombinant can serve as a spike-in control in protein lysate arrays. Purity is >95% by SDS-PAGE, endotoxin <0.1 EU/µg, so background from contaminants is minimal.

How should I store and handle PC-9 (Recombinant) to maintain activity and avoid degradation?

Store PC-9 (Recombinant) at -20°C in single-use aliquots. The supplied buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol — is formulated to stabilize the propeptide-bound mature form through freeze-thaw. Avoid repeated freeze-thaw cycles: even two additional cycles can measurably reduce LDLR-binding activity. Upon thaw, keep on ice and use within the same working session. Do not dilute to working concentration and re-freeze. Shelf life at -20°C in unopened aliquots is 12 months from date of receipt. For short-term use (≤48 hours), storage at 4°C in the original buffer is acceptable.

What starting concentration of PC-9 (Recombinant) should I use for an LDLR degradation assay in HepG2 cells?

A dose–response range of 0.5–10 µg/mL (approximately 7–140 nM) is recommended when adding PC-9 (Recombinant) to HepG2 cell culture media to drive LDLR lysosomal degradation. The EC50 for LDLR reduction in hepatic cell lines typically falls between 1–3 µg/mL under standard serum-containing conditions (5–10% FBS). Note that serum LDL and endogenous PCSK9 in FBS will compete; consider using delipidated or PCSK9-depleted serum for cleaner dose–response curves. Confirm LDLR downregulation by Western blot using a validated anti-LDLR antibody, and use RP-PC9 on the same blot to verify exogenous PCSK9 presence.