Anti-Cathepsin B Rabbit Polyclonal Antibody

Frequently Asked Questions

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

Cathepsin B is synthesized as a 339-amino acid prepropeptide (~37 kDa) that undergoes proteolytic maturation in the lysosome. On Western blots, you will typically observe the mature two-chain form as a predominant band at approximately 25-30 kDa, representing the heavy and light chains held together by disulfide bonds under non-reducing conditions. Under reducing conditions, you may see the individual chains around 25 kDa and 5 kDa, though the smaller chain often runs off standard gels. In some lysates, particularly those with active lysosomal processing, you may also detect the pro-form at 37-43 kDa depending on glycosylation state.

What starting dilution should I use for Cathepsin B Western blots?

We recommend starting at 1:1000 dilution for Western blot applications with this polyclonal antibody. Cathepsin B is relatively abundant in lysosome-rich tissues and most cultured cell lines, so 1:1000 typically provides strong signal without excessive background. If working with tissues where Cathepsin B is highly expressed—such as liver, kidney, or thyroid—you may achieve acceptable results at 1:2000 or even 1:5000. Conversely, if studying cells with low lysosomal content or following knockdown experiments, begin at 1:500 to ensure detection. Titrate against your specific sample type; overnight incubation at 4°C often improves sensitivity over one-hour room-temperature protocols.

Is this Cathepsin B antibody validated in mouse and rat samples?

This antibody is validated in human samples. Mouse and rat are listed as predicted cross-reactive species based on sequence homology in the immunogen region; human, mouse, and rat Cathepsin B share approximately 85-90% identity across the mature enzyme. We have not internally validated performance in rodent lysates, so if you are working with mouse or rat samples, we recommend confirming specificity with a positive control lysate known to express Cathepsin B and including a Cathepsin B-knockout or knockdown sample if available. Published literature shows substantial cross-reactivity for most polyclonal Cathepsin B antibodies across mammalian species, but titre optimization may be necessary.

What sample preparation preserves Cathepsin B signal best?

Cathepsin B is a lysosomal cysteine protease that remains active in mildly acidic conditions and can undergo autocatalytic processing if lysates are mishandled. Lyse cells or tissues in cold RIPA or similar buffer containing protease inhibitor cocktail; include E-64 or other cysteine protease inhibitors to prevent autodegradation and artifactual processing of the pro-form. Keep samples on ice and process quickly; freeze aliquots immediately if not running the same day. Avoid repeated freeze-thaw cycles, which promote aggregation and processing. For IHC applications, standard formalin fixation and paraffin embedding are compatible; antigen retrieval with citrate buffer (pH 6.0) at sub-boiling temperature typically yields optimal epitope exposure for this antibody.

Does Cathepsin B show both endopeptidase and exopeptidase activity, and does antibody binding interfere?

Cathepsin B is unusual among cathepsins in exhibiting both endopeptidase and carboxydipeptidase (exopeptidase) activity due to a unique occluding loop near its active site. This antibody is a polyclonal raised against a broad region of the protein and is intended for detection applications—Western blot—not for functional inhibition or activity assays. Antibody binding may sterically interfere with substrate access if used in co-immunoprecipitation followed by activity measurements, though this has not been systematically tested. For enzymatic assays, use the antibody for post-assay validation or localization studies rather than during the catalytic reaction itself.

What are good positive and negative controls for Cathepsin B experiments?

Positive controls should include lysates from lysosome-rich cell types such as HeLa, RAW264.7 macrophages, or primary hepatocytes, all of which express Cathepsin B constitutively. Thyroid tissue lysates are particularly strong positive controls given Cathepsin B's role in thyroglobulin processing. For negative controls, consider lysates from cells treated with Cathepsin B-targeting siRNA or CRISPR knockout lines if available; these confirm antibody specificity. Alternatively, preincubate the antibody with recombinant Cathepsin B protein as a blocking peptide competition assay. Be cautious interpreting complete absence, as Cathepsin B is widely expressed; reduced signal in knockdown samples is more realistic than complete loss.

Can I use this antibody for immunofluorescence to visualize lysosomal Cathepsin B?

Yes, this antibody is validated for Western blot; immunofluorescence validation is in progress. In fixed cells, expect punctate perinuclear staining consistent with lysosomal localization; co-staining with LAMP1 or LAMP2 provides excellent spatial confirmation. Use standard methanol or paraformaldehyde fixation followed by permeabilization with 0.1-0.5% Triton X-100. Start with a 1:100 to 1:200 dilution of primary antibody; lysosomal antigens are often abundant enough for robust signal at these dilutions. Under some stress conditions or in cancer cells, Cathepsin B relocalizes to the cytosol or secretory pathway, so diffuse staining does not necessarily indicate antibody failure but may reflect biological redistribution.

How should I store this Cathepsin B antibody and what is the shelf life?

Store the antibody at -20°C in the supplied glycerol-containing storage buffer; do not freeze in frost-free freezers, which cycle temperature. The 100 µg aliquot is stable for at least 12 months under these conditions based on our internal QC testing. For frequent use, prepare working aliquots (e.g., 10 µL volumes) to avoid repeated freeze-thaw, which can reduce titre and increase aggregation. If you must store diluted antibody, prepare it in PBS with 0.02% sodium azide and 1% BSA, keep at 4°C, and use within two weeks. Rabbit polyclonal antibodies are generally robust, but cysteine protease targets like Cathepsin B benefit from careful handling to preserve optimal performance across applications.