Cathepsin F (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant Cathepsin F on SDS-PAGE and Western blot?

The full-length human Cathepsin F precursor (UniProt Q9UBX1) is 484 amino acids, predicting an unmodified MW of ~52 kDa. Produced in HEK293 cells, this recombinant carries native-like N-linked glycosylation, so the apparent MW on reducing SDS-PAGE typically resolves at 55–60 kDa. Depending on lot-specific glycosylation efficiency, a faint secondary band near the mature processed form (~34 kDa) may appear. Run 50–100 ng per lane for a clean Coomassie reference. The Coomassie-stained gel certificate of analysis accompanies every lot.

Is this recombinant Cathepsin F the full-length precursor or the processed mature form?

This product is produced as the full-length precursor (residues 1–484, including signal peptide and propeptide) in HEK293 cells, with the propeptide functioning as an intramolecular inhibitor that keeps the enzyme largely latent at neutral pH. Activation to the mature ~34 kDa catalytic domain occurs autocatalytically under mildly acidic, reducing conditions (pH 4.5–5.5, 5–10 mM DTT or 1–5 mM TCEP). For activity assays, a brief activation step at pH 5.0 is recommended before diluting to assay pH. The zymogen form is appropriate for structural, binding, and immunological reference applications.

What fluorogenic substrate should I use to measure recombinant Cathepsin F activity?

Z-Phe-Arg-AMC (benzyloxycarbonyl-Phe-Arg-7-amido-4-methylcoumarin) is the standard fluorogenic substrate for Cathepsin F, with excitation/emission at 380/460 nm. A working concentration of 25–100 µM substrate in assay buffer (50 mM sodium acetate pH 5.0, 5 mM DTT, 1 mM EDTA) provides a linear signal window with 50–200 ng/mL of activated recombinant. Z-Arg-Arg-AMC is a secondary option for higher selectivity over Cathepsin B in mixed-substrate experiments. Confirm linearity at your detection gain before setting up inhibitor titration series.

What assay buffer conditions work best for Cathepsin F activity and IC50 measurements?

Cathepsin F is optimally active between pH 4.5 and 5.5, consistent with its lysosomal compartment. A standard activity buffer of 50 mM sodium acetate pH 5.0, 5 mM DTT, 1 mM EDTA at 37 °C gives reproducible Km/Vmax parameters. For IC50 determinations, pre-incubate enzyme with inhibitor for 15–30 min at pH 5.0 before substrate addition to allow equilibrium binding — cysteine cathepsin inhibitors (e.g., E-64 analogues) are time-dependent, and skipping pre-incubation inflates apparent IC50 values significantly. Verify that DMSO from inhibitor stocks does not exceed 0.5% v/v in the final assay.

What starting concentration of recombinant Cathepsin F should I use for a fluorometric activity assay?

A working range of 50–200 ng/mL activated recombinant Cathepsin F in assay buffer is appropriate for initial substrate Km characterisation using Z-Phe-Arg-AMC. Begin at 100 ng/mL and titrate down if the fluorescence signal exceeds the linear range of your plate reader within the first 5 minutes. Activation prior to use: dilute to 1 µg/mL in 50 mM sodium acetate pH 5.0, 5 mM DTT, incubate 10 min at 37 °C, then dilute to working concentration. Avoid diluting into the TPB storage buffer (Tris-HCl pH 7.5) immediately before assay — the neutral pH will suppress activity.

Can I use this recombinant Cathepsin F as a positive control antigen for Western blot with the matched antibody?

Yes — this is a primary validated use case. The matched antibody, RP-CathepsinF (rabbit polyclonal, /anti-cathepsin-f-rabbit-polyclonal-antibody), was raised and validated against recombinant Cathepsin F produced in the same HEK293 system, so epitope compatibility is confirmed rather than predicted. Load 20–50 ng of recombinant per lane alongside your cell lysate lanes; the antibody detects the ~55–60 kDa glycosylated precursor under reducing conditions. This pairing is particularly useful when you need to verify antibody performance in a new tissue or species context before committing sample lanes.

How much recombinant Cathepsin F should I load for a Western blot positive control lane?

For a standard ECL-based Western blot with RP-CathepsinF antibody, load 20–50 ng of recombinant Cathepsin F per lane. At 20 ng you should see a clean band at 55–60 kDa; increasing to 50 ng sharpens the signal if your secondary antibody or film sensitivity is limiting. Dilute the stock into 1× SDS sample buffer (reducing) and do not boil longer than 5 min — excessive heat can partially deglycosylate or aggregate the protein, producing anomalous smearing above 60 kDa. Running 20 ng alongside 30 µg of positive tissue lysate provides a useful intensity reference point.

How should I store and handle recombinant Cathepsin F to preserve activity over time?

The protein is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol and is stable at −20 °C in single-use aliquots for at least 12 months from the lot release date. Repeated freeze-thaw cycles degrade cysteine protease activity measurably — each cycle can reduce specific activity by 10–20% — so aliquot to your typical experiment volume before freezing. For short-term use (1–2 weeks), the protein is stable at 4 °C. Add fresh DTT or TCEP immediately before activity assays; the storage buffer does not contain reducing agent, and the catalytic cysteine oxidises at neutral pH without reductant present.