Cathepsin K (Recombinant)

Frequently Asked Questions

What molecular weight does recombinant Cathepsin K run at on SDS-PAGE and Western blot?

The mature processed form of human Cathepsin K (residues 115–329 after signal peptide and propeptide removal) has a predicted MW of ~23 kDa; however, N-linked glycosylation from HEK293 expression typically shifts the observed band to ~27–30 kDa under denaturing SDS-PAGE conditions. Under reducing conditions a single band is expected in that range at >90% purity. If you are running a non-reducing gel, note that the two conserved disulfide bonds will alter migration slightly. Plan your ladder range accordingly — a 10–50 kDa range covers both the glycosylated and deglycosylated forms.

What is the processing state of this recombinant Cathepsin K — is it the zymogen or the mature active form?

This product is supplied as the mature, active two-chain form of Cathepsin K, not the full-length zymogen. The 38-amino-acid signal peptide and the ~62-residue propeptide (which acts as an endogenous inhibitor at neutral pH) have been removed during HEK293 processing. What you receive is the catalytically competent mature enzyme corresponding to residues 115–329 of UniProt P43235. No additional activation step is required before use. This is especially relevant for activity assays — you can proceed directly from thaw to assay without preincubation with reducing agent alone, though DTT is still recommended to ensure active-site cysteine (Cys139) is fully reduced.

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

Cathepsin K efficiently cleaves type I collagen, fibrinogen, and thyroglobulin in vivo. For in vitro fluorogenic activity assays, Z-Leu-Arg-AMC (Cbz-Leu-Arg-7-amido-4-methylcoumarin) is the most widely used substrate for Cathepsin K, with Km values typically in the 5–20 µM range under acidic conditions. Z-Gly-Pro-Arg-AMC is also accepted. Avoid Z-Phe-Arg-AMC as the primary substrate — it is more selective for Cathepsin B and L and will underestimate Cathepsin K-specific activity. Monitor AMC release at Ex/Em 360/460 nm. Always include a no-enzyme blank and an E-64 inhibitor control to confirm cysteine protease-dependent signal.

What buffer conditions are optimal for Cathepsin K activity assays — pH, reducing agent, and temperature?

Cathepsin K has peak activity between pH 5.5 and 6.5, consistent with its lysosomal environment. A standard assay buffer of 50 mM sodium acetate pH 5.5, 5 mM DTT, 2.5 mM EDTA works well for fluorogenic substrate assays. DTT (or TCEP at 1–2 mM) is essential to maintain the catalytic Cys139 in the reduced, active state — omitting reducing agent will substantially reduce or abolish activity. Assay at 37°C for physiological relevance, or 25°C for slower, more controllable kinetics. Note that the storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol) is not the assay buffer; dilute the enzyme into activity assay buffer immediately before use.

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

For Z-Leu-Arg-AMC fluorogenic inhibitor screening, a Cathepsin K enzyme concentration of 1–5 nM in assay buffer (50 mM sodium acetate pH 5.5, 5 mM DTT, 2.5 mM EDTA) typically gives a robust signal window with substrate at 20–50 µM (well below saturating concentrations to keep conditions suitable for Ki determination). Pre-incubate enzyme with inhibitor for 15–30 minutes at 37°C before adding substrate to allow equilibration, particularly for slow-binding inhibitors such as odanacatib analogs. Run a substrate titration on your plate reader first to confirm linearity before committing compound libraries.

Can I use recombinant Cathepsin K as a positive control for Western blot with TPB's matched anti-Cathepsin K antibody?

Yes — this is a validated pairing. The matched antibody, SKU RP-CathepsinK (/anti-cathepsin-k-rabbit-polyclonal-antibody), is a rabbit polyclonal raised against human Cathepsin K and has been validated for Western blot using this recombinant protein as the positive control antigen in-house. Load 10–50 ng of REC-CathepsinK per lane alongside your cell lysates; the antibody reliably detects the ~27–30 kDa glycosylated band. Because TPB has validated this antibody–antigen pair from the same production batch, you get a predictable reference band without needing to source a separate standard — useful for antibody lot-to-lot comparisons and assay setup.

How much recombinant Cathepsin K should I load as a positive control for Western blot, and what band should I expect?

Load 10–50 ng per lane under reducing, denaturing conditions. At 10 ng, RP-CathepsinK detects a clear ~27–30 kDa band (glycosylated mature form) with minimal background on standard PVDF or nitrocellulose membranes after blocking with 5% non-fat milk in TBST. If your primary antibody concentration is at the lower end of the recommended range (e.g., 1:2000 dilution of RP-CathepsinK), loading 25–50 ng provides a stronger reference. Do not exceed 100 ng — overloading can cause band spreading and obscure endogenous signals in adjacent lysate lanes. Always run the recombinant in a dedicated lane rather than spiking into complex lysate.

How should I store and handle recombinant Cathepsin K after arrival to maintain activity — any freeze-thaw considerations?

Upon receipt, store at -20°C in the single-use aliquots as supplied. The storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol) stabilizes the enzyme during freeze-thaw, but cysteine proteases are particularly sensitive to oxidation of the active-site thiol. Avoid repeated freeze-thaw cycles — activity loss of >20% has been reported after three cycles for similar cysteine proteases. If you anticipate daily use over one to two weeks, keep a working aliquot at 4°C with freshly added DTT (1–2 mM) and use within 7 days. Do not dilute in buffers containing heavy metals or iodoacetamide, both of which irreversibly alkylate Cys139.