Anti-Cathepsin W Rabbit Polyclonal Antibody

Frequently Asked Questions

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

Cathepsin W migrates at approximately 42 kDa on reducing SDS-PAGE, corresponding to the full-length 376-amino acid zymogen. Because CTSW is synthesized with an N-terminal propeptide that undergoes proteolytic processing for activation, you may also observe lower molecular weight bands around 28-32 kDa representing the mature, processed form. The band pattern depends on your sample type: CD8+ T cells or NK cell lysates enriched for CTSW often show both pro- and mature forms, whereas samples with lower expression may show only the zymogen. Running a positive control lysate from activated cytotoxic lymphocytes helps distinguish specific signal from background.

Is this antibody validated for mouse Cathepsin W or only human?

This antibody has been validated with human samples. Mouse and rat cross-reactivity are predicted based on epitope homology but have not been experimentally confirmed in our hands. Human and mouse CTSW share approximately 78% sequence identity, so cross-reactivity is plausible, particularly if the epitope falls within a conserved region. If you are working with mouse samples, we recommend titrating the antibody starting at the 1:1000 dilution used for human, running a known-positive control such as activated mouse splenocytes or an NK cell line, and including a blocking peptide control if available to confirm specificity.

What positive control cell type should I use for Cathepsin W Western blots?

CD8+ cytotoxic T lymphocytes and natural killer cells are the most reliable positive controls, as CTSW expression is highly restricted to these immune effector populations. Activated human PBMCs, particularly after stimulation with IL-2 or anti-CD3/CD28, show robust CTSW expression. NK cell lines such as NK-92 or YT cells are also suitable. Negative controls should include non-lymphoid tissues or CD4+ T cells, which express little to no Cathepsin W. Whole spleen or lymph node lysates may show weak signal due to dilution by non-expressing cell types, so enriched lymphocyte populations yield clearer results.

Can I use this antibody for immunofluorescence to see Cathepsin W localization?

Yes, Triple Point antibodies are validated for immunofluorescence as well as Western blot and IHC. Given that Cathepsin W localizes primarily to the endoplasmic reticulum in resting cells, you should see a perinuclear reticular staining pattern in positive cells. In activated cytotoxic lymphocytes, CTSW may redistribute to secretory lysosomes or granules. Start with a 1:100 to 1:200 dilution for IF, as this is typically less sensitive than Western blot. Permeabilization is essential since CTSW is intracellular. Co-staining with an ER marker such as calnexin or PDI can help confirm the expected subcellular distribution in your system.

What dilution should I start with for Western blot of Cathepsin W?

We recommend starting at 1:1000 for Western blot, which has been validated with this antibody. This dilution typically provides clean signal with low background when using 20-30 µg of total protein from CTSW-expressing cell lysates. If your sample has low expression or you are working with whole tissue homogenates rather than purified lymphocytes, you may need to use a more concentrated dilution such as 1:500. Conversely, if background is high, titrate up to 1:2000. Blocking in 5% non-fat milk or BSA in TBST for one hour at room temperature works well for this rabbit polyclonal.

Does Cathepsin W have splice isoforms I need to account for?

UniProt lists a single canonical isoform for human Cathepsin W (P56202-1), and no functionally distinct splice variants have been well characterized in the literature. You should expect one major band corresponding to the 376-amino acid zymogen at approximately 42 kDa, plus possible lower molecular weight bands representing proteolytically processed mature forms. If you observe multiple high molecular weight bands, consider post-translational modifications such as glycosylation, which can affect migration. Unlike some cathepsins with tissue-specific variants, CTSW appears to be expressed as a single predominant form in cytotoxic lymphocytes, simplifying interpretation of Western blot and IHC data.

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

Store the antibody at -20°C in small aliquots to avoid repeated freeze-thaw cycles, which can reduce titer and increase aggregation. The 100 µg pack size is suitable for dividing into 10-20 µL aliquots depending on your planned experiments. Once thawed, an aliquot can be stored at 4°C with 0.02% sodium azide for up to one month for convenience during active experiments. Avoid prolonged storage in frost-free freezers due to temperature cycling. Properly stored aliquots typically retain activity for at least two years from the manufacture date. If you notice increased background or reduced signal over time, prepare a fresh aliquot rather than re-using older working stocks.

Why do I see low or no signal for Cathepsin W in my samples?

Cathepsin W has extremely restricted expression, limited almost exclusively to CD8+ T cells and NK cells. If you are using whole tissue lysates, unfractionated PBMCs, or non-lymphoid cell lines, CTSW protein may be below the detection threshold. Verify that your sample contains cytotoxic lymphocytes and consider enriching CD8+ or NK populations by magnetic sorting or density gradient. CTSW expression is also upregulated upon activation, so resting cells may show weak signal. Running a validated positive control such as activated NK-92 cells or IL-2-stimulated PBMCs in parallel confirms that the antibody is working and helps distinguish true absence from technical failure.