Furin (Recombinant)

Frequently Asked Questions

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

The predicted molecular weight of Furin based on its 794-amino-acid sequence is approximately 87 kDa. However, due to N-linked glycosylation added during expression in HEK293 cells, the apparent MW on SDS-PAGE typically runs between 95–110 kDa under reducing conditions. If you are using this as a positive control with our matched antibody RP-Furin, expect a diffuse band in that range — glycoprotein heterogeneity is the norm for native-like Furin and is not indicative of degradation or batch failure.

What processing form is this recombinant Furin — prodomain-intact or mature active enzyme?

This recombinant corresponds to the full-length sequence (residues 1–794), which includes the signal peptide, inhibitory prodomain, catalytic subtilisin-like domain, and P-domain. During HEK293 expression, autocatalytic prodomain removal occurs intramolecularly in the ER and trans-Golgi network, consistent with native Furin maturation. The supplied protein is therefore predominantly the mature, active form. If your experiment specifically requires prodomain-intact Furin (e.g., to study prodomain inhibition kinetics), confirm activity assay results at your target pH before assuming full processing.

What consensus sequence does recombinant Furin cleave and what are good model substrates for activity assays?

Furin cleaves the consensus motif RX(K/R)R↓, with the scissile bond C-terminal to the last Arg. For fluorogenic activity assays, the peptide substrate Boc-Arg-Val-Arg-Arg-AMC (also sold as pERTKR-AMC) is widely used and recommended as a starting point. Assay at 37°C in 100 mM HEPES pH 7.5, 1 mM CaCl₂, 1 mM β-mercaptoethanol. A typical enzyme concentration of 1–5 nM recombinant Furin is sufficient to generate a linear fluorescence signal over 30–60 minutes. Furin is strongly inhibited by decanoyl-RVKR-CMK, useful as a negative control.

What buffer conditions are optimal for recombinant Furin enzymatic activity assays?

Furin activity is calcium-dependent and pH-sensitive. Optimal conditions are 100 mM HEPES or MES pH 7.0–7.5, supplemented with 1 mM CaCl₂ and a mild reductant (0.5–1 mM β-mercaptoethanol or DTT). Activity drops significantly below pH 6.5 or above pH 8.0. The storage buffer for this product is 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol — dilute directly into your assay buffer to minimize glycerol carryover, keeping final glycerol below 1% to avoid enzyme inhibition at higher concentrations.

What starting enzyme concentration should I use for a recombinant Furin inhibitor IC50 assay?

For IC50 determinations, use 1–5 nM recombinant Furin with 100 µM Boc-RVRR-AMC or pERTKR-AMC substrate in 100 mM HEPES pH 7.5, 1 mM CaCl₂. At these concentrations the Km for typical tetrapeptide substrates is in the 50–200 µM range, so substrate is well below saturation — appropriate for competitive inhibitor studies following Cheng-Prusoff correction. Confirm linear reaction rate over your measurement window before adding inhibitor series. Avoid BSA as a carrier protein in the assay buffer, as it can non-specifically bind hydrophobic inhibitor compounds.

Can I use recombinant Furin as a Western blot positive control for the RP-Furin antibody?

Yes — this is a primary intended use. Recombinant Furin (REC-Furin) and the matched rabbit polyclonal antibody RP-Furin are validated together from the same production pipeline. For a positive control lane, load 50–200 ng of recombinant protein per lane on a 10% SDS-PAGE gel under reducing conditions. Expect a band at 95–110 kDa (glycosylated form). RP-Furin is validated for Western blot detection of this recombinant, making it reliable for antibody lot qualification, dilution optimization, and as a molecular weight reference alongside cell lysate lanes.

How much recombinant Furin should I load for a Western blot positive control and what dilution of RP-Furin antibody should I use?

Load 100 ng of REC-Furin per lane as a starting point; titrate down to 50 ng or up to 200 ng depending on your detection system sensitivity (ECL vs. fluorescent). For the matched antibody RP-Furin, a primary antibody dilution of 1:1,000–1:5,000 in 5% non-fat milk or BSA in TBST is a reasonable starting range for Western blot. Refer to the RP-Furin product page (/anti-furin-rabbit-polyclonal-antibody) for lot-specific titer data. Running the recombinant alongside cell lysate allows direct comparison of endogenous vs. recombinant band position.

How should I handle, dilute, and store recombinant Furin to maintain activity over time?

REC-Furin is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol as single-use aliquots. Store at -20°C; avoid repeated freeze-thaw cycles, which progressively reduce specific activity. On the day of use, thaw on ice and dilute to working concentration in assay buffer immediately before use — do not hold diluted enzyme at room temperature for more than 30 minutes. For long-term stability beyond 6 months at -20°C, adding 0.1% BSA as a carrier protein to low-concentration aliquots (< 50 µg/mL) helps prevent surface adsorption losses.