Cathepsin W (Recombinant)

Frequently Asked Questions

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

The full-length human Cathepsin W precursor (376 aa, UniProt P56202) has a predicted molecular weight of ~43 kDa. On reducing SDS-PAGE, our HEK293-expressed recombinant typically resolves at approximately 45–48 kDa due to N-linked glycosylation added by the mammalian expression system. If you observe a lower band (~37–40 kDa) under denaturing conditions, this likely reflects partial removal of the signal peptide and propeptide during processing. Run alongside a prestained ladder in the 35–55 kDa range. Purity is >95% by SDS-PAGE, so the dominant band should be unambiguous.

Is recombinant Cathepsin W supplied as the full-length precursor or the mature processed form?

The recombinant is produced in HEK293 cells, which support native-like signal peptide cleavage and propeptide processing. The predominant species in solution corresponds to the mature form following removal of the N-terminal signal peptide (residues 1–22) and propeptide region, yielding an active catalytic domain. The lot-specific certificate of analysis confirms the processed form by N-terminal sequencing or mass spectrometry. Because Cathepsin W is proposed to traffic through the endoplasmic reticulum rather than lysosomes, the HEK293 system more accurately recapitulates this processing compared with E. coli or baculovirus expression.

What substrates does Cathepsin W cleave and what fluorogenic substrate should I use for an activity assay?

Cathepsin W is a cysteine protease with a Cys–His–Asn catalytic triad that shows endopeptidase activity. For fluorogenic activity assays, Z-Phe-Arg-AMC (carbobenzoxy-phenylalanine-arginine-7-amino-4-methylcoumarin) is the most widely used substrate, with excitation/emission at 360/460 nm. Cathepsin W has also been reported to cleave Z-Arg-Arg-AMC, though with lower efficiency. Begin with 50–100 µM substrate and 0.1–1 µg recombinant per 100 µL reaction. Confirm activity is cysteine-protease-dependent by including 1–10 mM E-64 as a negative control inhibitor.

What is the optimal assay buffer for Cathepsin W activity and does it require activation before use?

Cysteine cathepsins require a reducing environment to maintain the active-site Cys in its thiol form. Use 50 mM sodium acetate pH 5.5–6.0, 2 mM EDTA, 5 mM DTT (or 2 mM TCEP) for maximal activity. Although the storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol) stabilizes the enzyme during storage, transfer to acidic assay buffer immediately before the experiment. Pre-incubate with DTT/TCEP at 37°C for 5–10 minutes before adding substrate to fully reduce the active-site cysteine. Avoid DMSO above 1% v/v, which can denature the enzyme.

What starting concentration of recombinant Cathepsin W should I use when determining inhibitor IC50 values?

For IC50 determinations, use the lowest enzyme concentration that gives a robust signal-to-noise ratio — typically 0.1–0.5 µg/mL (approximately 2–11 nM based on the ~45 kDa processed form) with 50–100 µM Z-Phe-Arg-AMC substrate in 50 mM sodium acetate pH 5.5, 2 mM EDTA, 5 mM DTT. Enzyme concentration should be kept well below the Km of your substrate to ensure the measured IC50 reflects true inhibitor potency rather than enzyme excess. Titrate enzyme across a 0.05–1 µg/mL range in a preliminary linearity experiment before committing inhibitor compound to the full concentration–response curve.

Can I use this recombinant Cathepsin W protein as a positive control for Western blot with your matched antibody?

Yes — this is a primary intended use. The matched antibody, SKU RP-CathepsinW (rabbit polyclonal, /anti-cathepsin-w-rabbit-polyclonal-antibody), was validated against this same recombinant protein in our laboratory. Loading 20–50 ng of recombinant Cathepsin W per lane on a 10–12% SDS-PAGE gel under reducing conditions gives a clean, strong band at ~45–48 kDa with RP-CathepsinW at 1:1,000–1:2,000 dilution. This positive control is particularly useful when probing cell lysates from CTL or NK cell lines where endogenous Cathepsin W expression may be low or variable.

How much recombinant Cathepsin W should I load for Western blot antibody validation alongside RP-CathepsinW?

For antibody validation lanes, 20–50 ng of recombinant per lane is sufficient to produce a clearly detectable band without saturating the signal, assuming standard ECL detection. If your cell lysate lanes contain 20–40 µg total protein, include a 30 ng recombinant lane as an adjacent reference. This directly confirms the antibody (RP-CathepsinW) is detecting the correct ~45–48 kDa species in your biological sample. Including a no-primary control alongside confirms specificity. The >95% purity of the recombinant means you will not see confounding secondary bands that could complicate interpretation.

How should I store and handle recombinant Cathepsin W to preserve activity, and what is the shelf life?

Store at -20°C in the single-use aliquots provided. The storage buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol — stabilizes the enzyme for at least 12 months at -20°C without significant loss of activity, provided freeze-thaw cycles are avoided. Do not store working aliquots at 4°C for more than 24–48 hours, as cysteine proteases are susceptible to oxidative inactivation. When thawing, place on ice rather than at room temperature and add DTT or TCEP to 1–2 mM immediately before use if proceeding to an activity assay. Carrier protein (0.1% BSA) can be added to dilute aliquots to reduce adsorptive losses at low concentrations.