Cathepsin A (Recombinant)

Frequently Asked Questions

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

The CTSA gene encodes a 480-amino-acid precursor (~54 kDa unmodified), but HEK293-expressed Cathepsin A undergoes N-linked glycosylation and proteolytic processing into a two-chain mature form (a ~32 kDa heavy chain and a ~20 kDa light chain linked by a disulfide bond). Under reducing SDS-PAGE conditions expect two bands near 32 and 20 kDa; under non-reducing conditions the disulfide-linked heterodimer migrates closer to 54–58 kDa. Glycosylation typically adds 4–8 kDa above theoretical mass, so exact migration can vary slightly by lot.

Is this recombinant Cathepsin A the mature processed form or the zymogen precursor?

The HEK293-expressed material is the mature, processed form. Cathepsin A is synthesized as a 54 kDa single-chain precursor that undergoes lysosomal cleavage to yield the active heavy-chain/light-chain heterodimer. Our recombinant is supplied as this processed, enzymatically active species — not the uncleaved zymogen — making it suitable for carboxypeptidase activity assays and chaperone-function studies without requiring an additional activation step.

What substrate and assay conditions should I use to measure recombinant Cathepsin A carboxypeptidase activity?

Cathepsin A (EC 3.4.16.5) is most commonly assayed using the fluorogenic substrate Z-Phe-Ala-AMC or the chromogenic substrate Cbz-Phe-Ala-pNA, both cleaved at the C-terminal amide or ester bond. A typical assay buffer is 100 mM sodium acetate pH 5.0, 150 mM NaCl — reflecting the acidic lysosomal optimum. Monitor fluorescence at Ex/Em 360/460 nm (AMC release) or absorbance at 405 nm. Start with 20–50 nM recombinant Cathepsin A and 200 µM substrate; titrate substrate from 25–500 µM to determine Km. Include 1 mM PMSF inhibition control to confirm serine-carboxypeptidase-dependent signal.

What buffer is recombinant Cathepsin A supplied in, and does it interfere with downstream assays?

The protein is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol. Glycerol at 10% is generally enzyme-protective and does not inhibit carboxypeptidase activity at typical working dilutions (1:10 to 1:50 into assay buffer). Note that Cathepsin A activity is optimal near pH 5.0; diluting into sodium acetate assay buffer drops glycerol carry-over below 1%, which is well tolerated. If studying chaperone interactions with β-galactosidase or NEU1, the neutral-pH storage buffer is preferable, as both partner enzymes are unstable below pH 6.5.

What starting concentration of recombinant Cathepsin A should I use for an in vitro chaperone protection assay with NEU1 or β-galactosidase?

Published protection assays (e.g., Bonten et al.) typically use a 1:1 to 5:1 molar ratio of Cathepsin A to partner enzyme (NEU1 or β-galactosidase) in a lysosomal-mimic buffer (50 mM sodium acetate pH 4.5–5.0, 150 mM NaCl) at 37 °C for 30–60 min. A practical starting point is 200–500 nM Cathepsin A with 100 nM partner enzyme. Confirm partner enzyme stability by measuring residual activity (β-galactosidase: MUG substrate, Ex/Em 365/450 nm; NEU1: 4-MU-NANA substrate) after incubation with and without Cathepsin A.

Can I use recombinant Cathepsin A as a positive control on Western blot with the RP-CathepsinA antibody?

Yes — this is a primary use case for pairing REC-CathepsinA with RP-CathepsinA. Load 50–100 ng of recombinant Cathepsin A per lane under reducing conditions; the heavy-chain (~32–36 kDa, glycosylated) and light-chain (~20 kDa) bands should both be detected by RP-CathepsinA. The antibody was raised against a recombinant antigen from the same expression platform, so epitope recognition is reliable. Use the recombinant lane alongside your cell or tissue lysate to confirm band identity and calibrate exposure. See /anti-cathepsin-a-rabbit-polyclonal-antibody for recommended antibody dilutions.

How much recombinant Cathepsin A should I load for Western blot if I'm validating the RP-CathepsinA antibody?

For antibody validation purposes, a two-point titration works well: load 25 ng and 100 ng of REC-CathepsinA on the same gel alongside your sample lysate. This demonstrates linearity and confirms the antibody detects both the ~32 kDa heavy chain and ~20 kDa light chain (reducing conditions). If running non-reducing conditions, expect a single heterodimeric band at ~54–58 kDa. RP-CathepsinA is validated for Western blot at the dilution ranges listed on the antibody page (/anti-cathepsin-a-rabbit-polyclonal-antibody); use a standard HRP-conjugated secondary against rabbit IgG.

How should I store and handle recombinant Cathepsin A to maintain activity, and what is its shelf life?

Upon receipt, spin briefly to collect contents, then aliquot into single-use volumes to avoid repeated freeze-thaw, which progressively denatures the disulfide-linked heterodimer and reduces carboxypeptidase activity. Store aliquots at -20 °C; do not store at 4 °C long-term. When thawing, keep on ice and use within 4 hours. Shelf life is 12 months from date of receipt under recommended storage. Dilutions in assay buffer should be prepared fresh; carrier protein (0.1% BSA) can be added to stabilize dilute working stocks below 10 nM. Avoid detergents other than 0.01% Tween-20 in working buffers.