Anti-Cathepsin K Rabbit Polyclonal Antibody

Frequently Asked Questions

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

Cathepsin K migrates as multiple bands corresponding to its processing states. The full-length proenzyme runs at approximately 37 kDa, while the mature active form appears at 27-29 kDa following proteolytic removal of the N-terminal propeptide in the lysosome. You may observe both forms depending on cell type and lysis conditions. Osteoclasts and cells with active lysosomal processing typically show the mature form predominantly. Glycosylation variants can cause minor shifts. Include a positive control lysate from osteoclast-like cells or cathepsin K-transfected cells to confirm band identity in your system.

What dilution should I start with for Western blot with this cathepsin K antibody?

We recommend starting at 1:1000 dilution for Western blot based on validation data. This dilution has been optimized for detecting endogenous cathepsin K in osteoclast lysates and overexpressed protein in transfected cell lines. If you are working with tissues or cell types that express cathepsin K at low levels, such as certain fibroblasts or macrophage subsets, you may need to use a more concentrated dilution around 1:500. Always titre the antibody against your specific sample type, as expression levels vary widely. Overnight incubation at 4°C often improves sensitivity compared to shorter room-temperature protocols.

Does this antibody cross-react with other cathepsins?

This polyclonal antibody was raised against cathepsin K-specific epitopes and shows no detectable cross-reactivity with closely related cathepsins (B, L, S, V) in our validation experiments using recombinant proteins and knockout cell lysates. Cathepsin K shares structural homology with other cysteine cathepsins, but sequence divergence in the immunogenic regions used provides specificity. If you are working with samples expressing multiple cathepsins at high levels, consider running a cathepsin K knockout or knockdown control alongside wild-type samples to confirm band specificity. This control is particularly useful in macrophages and dendritic cells, which co-express several cathepsin family members.

Is this cathepsin K antibody validated for mouse and rat samples?

Human cathepsin K is the validated species for this antibody. Mouse and rat are listed as predicted cross-reactive based on 87% and 86% sequence identity, respectively, in the epitope region. We have preliminary data showing detection of murine cathepsin K in osteoclast cultures, but formal validation across multiple mouse tissues is ongoing. If you are working with rodent samples, we recommend confirming specificity with a positive control such as bone marrow-derived osteoclasts differentiated with RANKL, which express high levels of cathepsin K. A cathepsin K knockout mouse lysate provides the most rigorous negative control.

What sample preparation works best for detecting cathepsin K in osteoclasts?

Osteoclasts express cathepsin K abundantly but compartmentalize it within lysosomes and secretory vesicles. Use RIPA or a Triton X-100-based lysis buffer with protease inhibitors to ensure efficient extraction from membrane-bound compartments. Avoid overly harsh conditions that denature the protein before transfer. For bone tissue samples, decalcification is necessary but can reduce antigenicity; EDTA-based decalcification preserves epitopes better than acid-based methods. If you are examining secreted cathepsin K in conditioned medium or resorption assays, concentrate the medium by TCA precipitation or ultrafiltration, as secreted enzyme concentrations are typically low.

Can I use this antibody for immunohistochemistry on bone sections?

Yes, this antibody is validated for Western blot; IHC validation is in progress and works well on bone tissue sections. Antigen retrieval is essential; citrate buffer (pH 6.0) heat-induced epitope retrieval typically gives optimal results on paraffin-embedded decalcified bone. Cathepsin K localizes strongly to osteoclasts lining resorption surfaces and appears as punctate lysosomal staining within the cytoplasm. You should observe minimal background in osteoblasts and bone lining cells. For frozen sections, brief fixation in 4% paraformaldehyde followed by permeabilization gives good signal without excessive background. Include a known positive region such as the endosteal surface of actively remodeling trabecular bone as an internal control.

How should I store this cathepsin K antibody to maintain stability?

Store the antibody at -20°C in single-use aliquots to avoid repeated freeze-thaw cycles, which can reduce titre and increase aggregation. The antibody is supplied in a glycerol-containing buffer that remains liquid at -20°C, allowing you to remove small volumes without thawing the entire stock if you choose not to aliquot. For short-term use over 2-3 weeks, storing at 4°C is acceptable. Avoid prolonged storage in frost-free freezers, as temperature cycling degrades antibody performance. Do not store diluted antibody; prepare working dilutions fresh in blocking buffer on the day of use for optimal results.

What positive and negative controls should I use when validating cathepsin K expression?

The most reliable positive control is lysate from RANKL-differentiated bone marrow macrophages or the RAW264.7 osteoclast-like cell line after differentiation, both of which express high levels of cathepsin K. Human osteosarcoma lines such as U-2 OS typically show low or absent expression and serve as useful negative controls. For genetic validation, cathepsin K knockdown using siRNA or CRISPR provides definitive proof of band identity. Tissue-level controls include sections of actively remodeling bone (positive) versus cartilage or muscle (negative). Thyroid tissue shows moderate cathepsin K expression in follicular epithelium and can serve as an additional positive control.