Anti-Cathepsin V Rabbit Polyclonal Antibody

Frequently Asked Questions

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

Cathepsin V is synthesized as a 334-residue preproenzyme. On reducing SDS-PAGE you should expect the proenzyme around 37-39 kDa and the mature, processed single-chain form near 27-29 kDa. A two-chain mature form with a heavy chain near 22 kDa and a light chain near 5 kDa has also been reported, though the light chain is typically lost on standard gels. Which species predominates depends on tissue and lysis conditions; lysosome-rich samples (thymus, testis, cornea, epidermis) tend to favour mature forms, while overexpression systems often retain more proenzyme.

Does this antibody cross-react with Cathepsin L (CTSL)?

Cathepsin V shares roughly 78% sequence identity with Cathepsin L, so cross-reactivity is a legitimate concern for any anti-CTSV reagent. This antibody is validated on human samples for CTSV detection, but cross-reactivity with CTSL has not been formally excluded. If you are working in a system that co-expresses both (many epithelial and immune tissues), we recommend running a CTSV knockdown or knockout control alongside a CTSL-only cell line to confirm specificity in your hands. Mass spec confirmation of the immunoreactive band is the cleanest orthogonal validation.

What is a good starting dilution for Western blot?

Start at 1:1000 in 5% non-fat milk or BSA in TBST, which is the validated dilution on this lot. For low-expression samples, titre between 1:500 and 1:1000; for thymus or testis lysates with high endogenous CTSV, 1:2000 may give cleaner background. Load 20-40 µg total protein, transfer at standard conditions for a ~30 kDa target (no extended transfer needed), and use a 4-hour room-temperature or overnight 4°C primary incubation. Image acquisition should be exposure-matched across samples to avoid misreading proenzyme/mature ratios.

Which positive control lysates work best for Cathepsin V?

Tissue-wise, human thymus and testis lysates show the highest endogenous CTSV expression and are the most reliable positive controls. Cornea and epidermis (or primary keratinocytes) are also strong but less practical to source. Among cell lines, MCF-7, HeLa, and HEK293 express detectable CTSV, with MCF-7 generally giving the clearest mature-band signal. For a negative or low-expression reference, most lymphoid cell lines and unstimulated PBMCs show minimal CTSV. A CTSV-transfected HEK293 lysate is useful as a band-identity anchor if you have not run the target before.

Is this antibody validated for species other than human?

Validated reactivity is human only. Reactivity in mouse and rat is not confirmed; the immunogen region shares partial identity with murine Cathepsin L (mice do not have a direct CTSV orthologue, since CTSV is considered a human/primate-specific paralogue of CTSL). We would not recommend this clone for rodent work without prior in-house validation against a CTSL knockout background. For non-human primate samples, reactivity is predicted based on sequence conservation but has not been formally tested.

What sample preparation is best for detecting mature Cathepsin V?

Because CTSV is lysosomal, standard RIPA lysis at neutral pH can drive partial autoactivation and degradation during extraction. Lyse on ice in the presence of broad cysteine protease inhibitors (E-64 at 10 µM is preferable to leupeptin alone, which CTSV can tolerate). Avoid extended sonication and keep samples cold. For preserving the proenzyme/mature ratio, snap-freeze tissue and lyse directly into Laemmli buffer with 50 mM DTT. Reducing conditions are required to resolve the mature single-chain form; non-reducing gels will give ambiguous banding due to internal disulfides.

Can I use this antibody for IHC on FFPE sections?

Yes, the antibody is validated for IHC on formalin-fixed paraffin-embedded human tissue. Heat-induced epitope retrieval in citrate buffer (pH 6.0) for 20 minutes is the standard starting condition; Tris-EDTA (pH 9.0) can be tried if signal is weak. A starting dilution range of 1:100-1:400 is reasonable, with titration recommended per tissue. Expect granular cytoplasmic staining consistent with lysosomal localization, strongest in thymic epithelial cells, testicular germ cells, and corneal epithelium. Include a no-primary control and, where possible, a tissue known to be CTSV-low (e.g., liver) as a negative reference.

How should the antibody be stored and aliquoted?

Supplied as 100 µg of rabbit polyclonal IgG in PBS with 0.02% sodium azide and 50% glycerol. On receipt, store at -20°C for long-term use; the glycerol formulation prevents freezing at -20°C, so aliquoting is not strictly required, but we recommend splitting into 10-20 µL working aliquots if you anticipate frequent access. Avoid repeated freeze-thaw cycles (more than 5 cycles can reduce titre). The working aliquot in current use can be kept at 4°C for up to one month. Do not store diluted antibody; prepare working dilutions fresh on the day of use.