Anti-Cathepsin H Rabbit Polyclonal Antibody

Frequently Asked Questions

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

Mature cathepsin H typically appears at approximately 28 kDa on Western blots, corresponding to the processed, active form of the enzyme after removal of the signal peptide and propeptide. You may also detect pro-cathepsin H at approximately 37 kDa, depending on your cell type and lysis conditions. The relative intensity of these bands reflects the balance between zymogen and mature enzyme in your sample. Lysosome-enriched fractions or samples treated with lysosomal protease inhibitors during lysis may show a stronger pro-form band. Both bands represent legitimate cathepsin H species; their ratio provides information about proteolytic processing status in your system.

Does this antibody detect cathepsin H in monkey or mouse samples?

This antibody has been validated experimentally against human cathepsin H. Cross-reactivity with non-human primate species, including cynomolgus and rhesus monkey, is predicted based on high sequence homology in the epitope region, but we have not formally validated it in those systems. Mouse cathepsin H shares lower sequence identity with human; cross-reactivity is uncertain and should be empirically tested if you plan to use murine samples. If you require validated mouse reactivity, contact us to discuss whether species-specific validation data have become available since initial product release or whether an alternative clone may better suit your needs.

What starting dilution should I use for Western blot with this cathepsin H antibody?

Begin with a 1:1000 dilution in your blocking buffer for Western blot, which is the validated starting point for this antibody. Cathepsin H is moderately abundant in most lysate preparations, so this dilution typically yields clean signal without excessive background. If your initial blot shows weak signal, you can increase concentration to 1:500. Conversely, if background is problematic or you are working with lysosome-enriched fractions where cathepsin H is highly concentrated, titrate toward 1:2000. Optimal dilution depends on your expression level, transfer efficiency, and detection system; always include a lysate known to express cathepsin H as a positive control during optimization.

How should I prepare cell lysates to preserve cathepsin H for Western blot detection?

Lyse cells in a neutral-pH buffer containing protease inhibitors to prevent cathepsin H autodegradation and preserve both the pro-form and mature enzyme. Standard RIPA buffer works well, but ensure your protease inhibitor cocktail includes cysteine protease inhibitors such as E-64 or leupeptin, since cathepsin H is itself a cysteine protease. Avoid prolonged lysis at room temperature; keep samples on ice and process quickly. If your goal is to assess the mature, active form specifically, do not include cysteine protease inhibitors, but be aware this may lead to partial degradation. For subcellular fractionation studies, enrich lysosomal fractions to maximize signal, as cathepsin H is primarily localized to this compartment.

Can I use this antibody for immunohistochemistry or immunofluorescence on tissue sections?

Yes, This antibody is validated for Western blot; immunohistochemistry/immunofluorescence validation is in progress applications in addition to Western blot. Cathepsin H localizes predominantly to lysosomes, so expect punctate cytoplasmic staining in positive cells. Antigen retrieval is typically required for formalin-fixed, paraffin-embedded tissues; citrate buffer (pH 6.0) heat-induced epitope retrieval is a reasonable starting protocol. For immunofluorescence on cultured cells, standard methanol or paraformaldehyde fixation followed by permeabilization with Triton X-100 should preserve epitope accessibility. Dilution will need optimization for your specific system, but starting ranges of 1:100 to 1:200 are typical for IHC. Co-staining with a lysosomal marker such as LAMP1 can confirm compartment-specific localization.

What positive control lysate should I use to confirm this cathepsin H antibody is working?

HeLa or HEK293 whole-cell lysates serve as reliable positive controls, as cathepsin H is constitutively expressed in these commonly used cell lines. Lysates from macrophages or dendritic cells are also excellent positive controls given the role of cathepsin H in antigen processing; these cells typically show robust expression. Tissue lysates from liver or kidney, both of which contain abundant lysosomes, are suitable alternatives. Load 20–30 µg total protein per lane. If you see no signal with a validated positive control, troubleshoot your transfer efficiency, blocking conditions, and antibody dilution before concluding the antibody has failed. Include a molecular weight marker that spans the 25–40 kDa range to confirm band identity.

Does cathepsin H have isoforms or splice variants I need to account for in my experiment?

Cathepsin H is encoded by a single gene (CTSH) and does not have well-characterized splice variants that produce functionally distinct isoforms. The primary species you will encounter are the inactive pro-enzyme (approximately 37 kDa) and the mature, active form (approximately 28 kDa), which result from post-translational proteolytic processing rather than alternative splicing. The mature enzyme includes an unusual N-terminal mini-chain that remains disulfide-bonded to the catalytic domain, conferring aminopeptidase activity. This structural feature is consistent across samples and does not represent an isoform. If you observe additional bands, consider the possibility of non-specific binding, degradation, or cross-reaction with other cathepsins; use lysate from CTSH-knockout cells as a negative control if available.

How should I store this cathepsin H antibody and how long will it remain stable?

Store the antibody at –20°C in its original formulation; avoid repeated freeze-thaw cycles, which can reduce titer and increase aggregation. For routine use, prepare small aliquots (e.g., 10–20 µL) immediately upon receipt and store them separately so you thaw only what you need for each experiment. If you use the antibody frequently, a working aliquot can be held at 4°C with 0.02% sodium azide as preservative for several weeks without significant loss of activity. Under proper storage at –20°C, polyclonal antibodies typically retain reactivity for at least two years from the date of receipt. Check for visible precipitate before use; if present, centrifuge briefly at 10,000 × g and use the supernatant.