PC-4 (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant PC-4 (PCSK4) on SDS-PAGE?

Full-length human PCSK4 has a predicted molecular weight of ~83 kDa, but the recombinant protein produced in HEK293 cells typically migrates at 90–100 kDa under denaturing conditions due to N-linked glycosylation. The mature, autocatalytically processed form — generated after removal of the prodomain — runs at approximately 70–75 kDa. Depending on your expression construct and activation state, you may observe both species. Our SDS-PAGE QC consistently shows a predominant band in the 90–100 kDa region at >90% purity, with the mature form visible upon activation.

Which processing form of PCSK4 is present in this recombinant — zymogen or mature enzyme?

This preparation is produced in HEK293 mammalian cells, which support the autocatalytic zymogen-to-mature-form processing that is essential for PCSK family activation. The product is supplied predominantly as the active mature form following prodomain removal, retaining the catalytic subtilisin-like domain. HEK293 expression is specifically chosen over bacterial or insect-cell systems because the required folding, disulfide bond formation, glycosylation, and intracellular processing steps depend on intact mammalian cellular machinery. Residual zymogen may be present as a minor species; the active fraction is confirmed by fluorogenic substrate assay prior to release.

What substrates does PC-4 (PCSK4) cleave and how do I set up a fluorogenic activity assay?

PCSK4 cleaves after paired basic residue motifs (canonical -R/K-X-X-R↓- sites). For fluorogenic assays, peptide substrates such as Boc-RVRR-AMC or pGlu-RTKR-MCA are well-characterised starting points. Set up reactions in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM CaCl₂, 0.1% Triton X-100 at 37°C, monitoring AMC release at Ex/Em 360/460 nm. A typical starting enzyme concentration is 5–20 nM with substrate at 10–200 µM to bracket Km. Include a heat-inactivated control to confirm signal is enzyme-dependent. Adjust pH between 7.0 and 8.0 to assess pH-rate profiles.

What buffer is PC-4 recombinant supplied in and can I dilute it directly into my assay?

The protein is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol. This storage buffer is compatible with most enzymatic assay systems and can be diluted directly into your reaction mix without dialysis for most applications. If your assay requires low glycerol (<0.5%), dilute the enzyme to working concentration just before use to minimise activity loss from glycerol dilution. Avoid diluting below 0.1 µg/µL in the absence of a carrier protein — add 0.05% BSA to the dilution buffer to prevent surface adsorption losses. Do not introduce reducing agents such as DTT, as these can disrupt critical disulfide bonds.

How should I store recombinant PC-4 and what is the shelf life after thawing?

Store at -20°C in the single-use aliquots provided. Repeated freeze-thaw cycles measurably reduce enzymatic activity; even two additional cycles can result in >30% activity loss for serine endoproteases of this class. Once thawed, keep the aliquot on ice and use within 8 hours. Do not refreeze. For extended working-stock use, prepare further dilutions in assay buffer containing 0.05% BSA and store these at 4°C for no more than 24–48 hours. Shelf life is 12 months from receipt at -20°C when handled correctly. Check the CoA for the activity benchmark specific to your lot.

Can I use recombinant PC-4 as a positive control for Western blot with the matched RP-PC4 antibody?

Yes — this is one of the primary intended uses. The matched rabbit polyclonal antibody RP-PC4 (see /anti-pc-4-rabbit-polyclonal-antibody) was raised and validated against PCSK4 in the same laboratory, ensuring antigen–antibody compatibility. Load 5–20 ng of recombinant PC-4 per lane alongside your cell lysate samples. You should observe a strong band at 90–100 kDa (glycosylated full-length form) and potentially a 70–75 kDa mature-form band. This provides an unambiguous size reference and confirms antibody specificity, particularly useful when running samples from tissues with low endogenous PCSK4 expression.

How much recombinant PC-4 should I load for a Western blot positive control lane?

For most chemiluminescent Western blot setups using RP-PC4 at a 1:500–1:2000 primary antibody dilution, 10–20 ng of recombinant PC-4 per lane produces a robust, non-saturating signal. If you are optimising antibody concentration or comparing sensitivity between detection systems (ECL vs. fluorescent secondary), titrate from 5 ng to 50 ng across replicate lanes. Load the recombinant in Laemmli sample buffer with 5% β-mercaptoethanol and boil for 5 minutes. The denatured, reduced form resolves cleanly at 90–100 kDa and serves as a reliable benchmark for antibody lot-to-lot consistency validation.

How do I determine the IC50 of a PCSK4 inhibitor using this recombinant in a biochemical assay?

Use a fixed enzyme concentration of 5–10 nM PC-4 recombinant and a fluorogenic substrate (e.g., Boc-RVRR-AMC) at or near the apparent Km (~50–100 µM as a starting estimate) to ensure the assay is sensitive to competitive inhibitors. Pre-incubate enzyme with inhibitor for 15–30 minutes at room temperature before substrate addition to allow equilibrium binding. Measure initial velocity (linear phase, first 5–10 minutes) across 8–10 inhibitor concentrations spanning 3 log units. Plot vi/v0 versus [I] and fit to a four-parameter logistic model. Include a PCSK inhibitor reference compound (e.g., dec-RVKR-CMK) as an internal IC50 benchmark across runs.