Anti-Cathepsin S Rabbit Polyclonal Antibody

Frequently Asked Questions

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

Cathepsin S migrates as multiple bands reflecting its proteolytic maturation. The full-length zymogen (procathepsin S) runs at approximately 37-38 kDa. Following N-terminal propeptide cleavage in the lysosome, the mature active form appears at 24-28 kDa, which is the predominant species in most lysates from antigen-presenting cells. You may observe both forms depending on cell type and activation state. Glycosylation can add 2-3 kDa heterogeneity. Reducing conditions are standard; cathepsin S is a monomeric enzyme and does not require non-reducing gels for detection.

What dilution should I start with for Western blot and IHC with this cathepsin S antibody?

For Western blot, begin at 1:1000 dilution in 5% non-fat milk or BSA in TBST, which is our validated starting point for most human lysates. For IHC on formalin-fixed paraffin-embedded tissue, a range of 1:100 to 1:500 typically works after antigen retrieval in citrate buffer, pH 6.0. Expression varies considerably by cell type—cathepsin S is abundant in professional antigen-presenting cells such as dendritic cells, macrophages, and B cells, but low in most epithelial tissues. Titre the antibody against your specific sample type and fixation protocol to optimize signal-to-noise.

Is this antibody specific for cathepsin S or does it cross-react with other cathepsins?

This polyclonal was raised against a cathepsin S-specific immunogen and shows no detectable cross-reactivity with cathepsin L, K, or B in our validation panel, despite partial sequence homology among cysteine cathepsins. However, as with any polyclonal reagent, we recommend including a cathepsin S knockout lysate or siRNA knockdown control in your first experiment to confirm specificity in your biological system. Cathepsin S shares approximately 60% identity with cathepsin L in the catalytic domain, so orthogonal validation is good practice when interpreting expression patterns or colocalization studies.

Will this cathepsin S antibody work in mouse and rat samples?

Cross-reactivity with mouse and rat cathepsin S is predicted based on epitope homology but not yet validated in-house. Human and mouse cathepsin S share approximately 86% amino acid identity, and human-rat homology is similar. Several customers have reported successful detection in murine macrophage and dendritic cell lysates at 1:500 to 1:1000 dilution, but we advise running a positive control—such as LPS-stimulated bone marrow-derived dendritic cells—alongside your samples during optimization. If working exclusively with rodent models, request lot-specific data or plan a pilot Western to confirm signal before committing to large-scale experiments.

What sample preparation is recommended for detecting cathepsin S in cell lysates?

Use standard RIPA or NP-40 lysis buffer supplemented with protease inhibitor cocktail to preserve cathepsin S integrity. Because cathepsin S is a lysosomal protease, avoid freeze-thaw cycles that can cause lysosomal rupture and autocatalytic degradation. Process lysates on ice and snap-freeze aliquots in liquid nitrogen if long-term storage is required. For subcellular fractionation studies, be aware that cathepsin S is secreted by some immune cells and traffics through the endosomal-lysosomal pathway. Expected expression is highest in monocyte-derived cells, splenic macrophages, and microglia; low or absent in fibroblasts and most non-immune cell lines.

Can I use this antibody to detect extracellular or secreted cathepsin S?

Yes, but with caveats. While cathepsin S is primarily lysosomal, it is secreted by activated macrophages, dendritic cells, and some tumor cells, particularly under inflammatory conditions or in the tumor microenvironment. If detecting secreted cathepsin S in conditioned media or serum by Western blot, concentrate samples by TCA precipitation or ultrafiltration; secreted levels are typically 10- to 50-fold lower than intracellular. For ELISA-based quantification of soluble cathepsin S, a matched-pair sandwich immunoassay is more sensitive. This polyclonal will recognize both pro- and mature forms in extracellular compartments.

What positive and negative controls should I include when using this cathepsin S antibody?

Positive controls with robust cathepsin S expression include THP-1 cells differentiated with PMA, human peripheral blood monocyte-derived dendritic cells, or Raji B-cell lysates. Murine RAW 264.7 macrophages are a reasonable rodent positive control if cross-reactivity is confirmed. For negative controls, HEK293T or NIH-3T3 fibroblasts express negligible cathepsin S. The most rigorous specificity control is a lysate from cathepsin S-knockout cells or siRNA-treated samples run in parallel. Given cathepsin S upregulation in inflammation, consider stimulating cells with IFN-gamma or LPS to amplify signal during optimization.

How should I store this cathepsin S antibody and what is the shelf life?

Store the antibody at -20°C in small aliquots to avoid repeated freeze-thaw cycles, which can reduce titre and increase background. The antibody is supplied in PBS with glycerol and 0.02% sodium azide as preservative. Under these conditions, stability is typically greater than 12 months from date of receipt. For long-term storage beyond one year, keep at -80°C. Once thawed, an aliquot can be held at 4°C for up to one month if 0.02% azide is present. Do not store diluted antibody in working solutions for more than one week; prepare fresh dilutions for best results.