Cathepsin O (Recombinant)

Frequently Asked Questions

What molecular weight does recombinant Cathepsin O run at on SDS-PAGE or Western blot?

The full-length human CTSO precursor (UniProt P43234) is 321 amino acids with a predicted unprocessed MW of ~37 kDa. On reducing SDS-PAGE, the HEK293-expressed recombinant typically migrates at approximately 38–42 kDa due to N-linked glycosylation added by the mammalian expression system. Under denaturing WB conditions you may observe a diffuse or doublet band in this range reflecting glycoform heterogeneity. If you need a sharp, defined band for ladder calibration, treat the sample with PNGase F prior to electrophoresis to collapse glycoforms to the ~37 kDa core.

What processing form is this recombinant Cathepsin O — full-length precursor or mature active enzyme?

This recombinant is produced as the mature, catalytically active form following propeptide removal. HEK293 cells support the autocatalytic or trans-processing that cleaves the N-terminal propeptide (roughly residues 1–113 of the precursor), yielding the active enzyme domain. You should not expect a propeptide band in reducing SDS-PAGE. If your experimental model involves tracking propeptide processing as a readout, you would need to separately express the zymogen form — this product is not appropriate for that application.

What substrates does Cathepsin O cleave, and which fluorogenic peptide should I use in an activity assay?

Cathepsin O is a cysteine protease with a preference for substrates bearing hydrophobic residues at the P2 position, similar to other papain-family cathepsins. The fluorogenic substrate Z-Phe-Arg-AMC (carbobenzoxy-Phe-Arg-7-amino-4-methylcoumarin) is a well-established choice for initial activity confirmation at 1–10 µM substrate concentration. Release of AMC is monitored by excitation at 380 nm / emission at 460 nm. Cathepsin L also cleaves this substrate, so include a cathepsin-selective inhibitor (e.g., E-64 at 10 µM as a negative control) to confirm cysteine-protease-dependent signal.

What buffer conditions should I use for a Cathepsin O activity assay to approximate lysosomal pH?

For activity assays, use 50 mM sodium acetate, 1 mM EDTA, 2–5 mM DTT, pH 5.5. Pre-activate the enzyme by incubating in assay buffer with DTT for 10–15 minutes at 37°C before adding substrate — cysteine proteases require a reducing environment to maintain the active-site thiol in its reduced state. The storage buffer supplied (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol) maintains stability at -20°C but is not optimal for catalytic activity; dilute into pH 5.5 acetate buffer immediately before use. Avoid DMSO above 1% (v/v) in inhibitor dilutions.

What starting concentration of recombinant Cathepsin O should I use for an IC50 inhibitor assay?

A starting point of 5–20 nM recombinant Cathepsin O in the assay well is typical for IC50 determinations, keeping enzyme concentration well below the Ki of your inhibitor to avoid stoichiometric-binding artifacts. Use Z-Phe-Arg-AMC at a concentration bracketing the Km (empirically determined, but 10 µM is a reasonable starting estimate). Run an enzyme titration curve first to identify the linear range of AMC release over your assay window (30–60 min at 37°C, pH 5.5). For tight-binding inhibitors, drop enzyme to 1–2 nM and use Morrison's tight-binding IC50 equation.

Can I use Cathepsin O (Recombinant) as a positive control antigen for Western blot with the matched TPB antibody?

Yes — this recombinant is the recommended positive control for use with RP-CathepsinO (Triple Point Biologics rabbit polyclonal anti-Cathepsin O antibody; see /anti-cathepsin-o-rabbit-polyclonal-antibody). Both were developed in the same lab from the same antigen, so epitope compatibility is confirmed rather than predicted. Load 20–50 ng of recombinant Cathepsin O per lane alongside your cell lysate samples. The antibody is validated for Western blot, so you should see a clean band in the 38–42 kDa region. This is particularly useful when working with cell lines expressing low endogenous CTSO.

How much recombinant Cathepsin O should I load as a Western blot positive control to get a clean band?

Load 25–50 ng per lane as a starting point when using RP-CathepsinO at a 1:1,000–1:2,000 primary antibody dilution. This gives a clearly visible band without overwhelming the lane or cross-contaminating adjacent sample wells by diffusion. If you are optimizing antibody concentration, run a 2-fold dilution series of the recombinant (10, 25, 50, 100 ng) in parallel. Because HEK293-expressed material retains native glycosylation, the band position will closely match endogenous CTSO from human tissue or cell lysate, making it a reliable size reference as well as a signal control.

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

Recombinant Cathepsin O is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol and shipped on dry ice. Upon receipt, aliquot immediately into single-use volumes and store at -20°C; avoid repeated freeze-thaw cycles, which progressively denature cysteine proteases. Under these conditions, activity is stable for at least 12 months from the date of manufacture (see CoA for lot-specific expiry). Do not dilute the stock into water or low-salt buffers for long-term storage — the glycerol and salt are critical cryoprotectants. Prepare working dilutions in pH 5.5 acetate assay buffer immediately before each experiment.