Anti-Cathepsin Z Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight band should I expect for Cathepsin Z on a Western blot?

Cathepsin Z is synthesized as a 303-amino acid precursor of approximately 34 kDa. After proteolytic removal of the propeptide during lysosomal maturation, the mature enzyme runs at approximately 28-30 kDa on reducing SDS-PAGE. You may observe both forms depending on cell type and lysis conditions. Lysosomal-rich samples or those treated with lysosomal protease inhibitors often show the precursor band more prominently. If you see only high-molecular-weight bands above 40 kDa, consider incomplete reduction or antibody aggregation. Start with 1:1000 dilution in 5% non-fat milk in TBST for initial optimization.

Is this antibody validated for mouse and rat samples or only human?

This antibody has been validated against human Cathepsin Z. Mouse and rat cross-reactivity is predicted based on sequence homology but not experimentally confirmed by Triple Point Biologics. Human and mouse CTSZ share approximately 89% amino acid identity, rat approximately 88%. If working with rodent samples, plan to validate the antibody yourself using a known positive control lysate such as kidney or spleen homogenate, where Cathepsin Z is expressed at detectable levels. Start with the recommended 1:1000 dilution and include a human positive control on the same blot for comparison.

Does Cathepsin Z have alternatively spliced isoforms I need to account for?

The primary Cathepsin Z transcript (UniProt Q9UBR2) does not have widely characterized alternatively spliced isoforms that alter the coding sequence significantly. The enzyme exists primarily as a single gene product that undergoes post-translational processing from the 34 kDa zymogen to the 28-30 kDa mature form. Tissue-specific glycosylation may cause minor mobility shifts on gels. If you observe multiple bands in the 28-34 kDa range, these typically represent the propeptide cleavage intermediates rather than splice variants. For experiments focused on enzyme maturation, consider comparing lysates from different subcellular fractions.

What positive control lysate should I use for Cathepsin Z Western blots?

Cathepsin Z is broadly expressed but shows higher levels in immune tissues and kidney. Human or mouse spleen lysate, macrophage cell lines such as THP-1 or RAW 264.7, or kidney homogenates serve as reliable positive controls. Hela and HEK293 cells express Cathepsin Z at moderate levels and are convenient if already in use. For negative controls, recombinant expression systems without lysosomal trafficking may show reduced mature enzyme. Avoid over-loading protein; 20-30 µg total lysate per lane is usually sufficient. Include a lane with cathepsin B or L lysate to confirm specificity if cross-reactivity is a concern.

Can I use this antibody for immunofluorescence to visualize lysosomal localization?

Triple Point Biologics antibodies are validated for Western blot and immunohistochemistry; immunofluorescence protocols have not been specifically optimized for this Cathepsin Z antibody but the format is supported across the product line. For IF experiments, start with a 1:100 to 1:200 dilution in blocking buffer. Because Cathepsin Z localizes primarily to lysosomes, co-staining with LAMP1 or LysoTracker provides useful confirmation of specificity. Permeabilize cells with 0.1-0.2% Triton X-100 and fix with 4% paraformaldehyde. Expect punctate perinuclear staining consistent with lysosomal distribution. Validate with a blocking peptide if background is high.

Should I include protease inhibitors when preparing lysates for Cathepsin Z detection?

Yes. Cathepsin Z is a cysteine protease that can undergo autolysis and may be degraded by other lysosomal proteases during lysis. Include a cysteine protease inhibitor such as E-64 (10-20 µM) or a broad-spectrum cocktail containing cysteine protease inhibitors in your lysis buffer. Avoid freeze-thaw cycles, which can disrupt lysosomes and activate cathepsins. Lyse cells on ice and clarify lysates promptly by centrifugation. If studying the propeptide-to-mature enzyme ratio, consider comparing samples lysed at neutral pH with and without inhibitors to assess processing. EDTA or EGTA may be included to chelate metalloproteases without affecting Cathepsin Z activity.

How should I store the antibody and how long is it stable after reconstitution?

The antibody is supplied as 100 µg and should be stored at -20°C or -80°C. Avoid repeated freeze-thaw cycles, which denature immunoglobulins and reduce titer. Upon receipt, consider making single-use aliquots in storage buffer containing 50% glycerol, which remains liquid at -20°C and permits direct pipetting without thawing. If stored as recommended without freeze-thaw, polyclonal antibodies typically retain activity for 12-24 months. For working stocks, diluted antibody in TBST with 0.02% sodium azide can be stored at 4°C for one week. Do not store diluted antibody in milk or BSA for more than 24 hours due to bacterial growth risk.

What is the epitope or immunogen used to generate this Cathepsin Z antibody?

This rabbit polyclonal antibody was raised against a recombinant fragment or peptide corresponding to human Cathepsin Z. The specific epitope has not been mapped, which is typical for polyclonal sera. Because the antibody recognizes both precursor and mature forms on Western blots, the immunogen likely includes sequences outside the propeptide region. For experiments requiring epitope-specific detection, such as distinguishing active-site mutants or monitoring propeptide cleavage, consider using the antibody in combination with a C-terminal or N-terminal specific reagent. Contact Triple Point Biologics directly if your experimental design requires detailed immunogen information for a custom application.