Anti-CPVL Rabbit Polyclonal Antibody

Frequently Asked Questions

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

The unprocessed CPVL polypeptide is 476 amino acids with a calculated mass near 54 kDa. In practice, the protein is N-glycosylated and proteolytically processed during transit through the secretory pathway to lysosomes, so the apparent migration on SDS-PAGE typically falls between 54 and 60 kDa for the proform, with smaller mature fragments sometimes visible after lysosomal maturation. PNGase F treatment will collapse the upper band. If you see only a ~50 kDa species in lysosomal fractions, that is consistent with the processed mature enzyme rather than nonspecific signal.

Which cell types or tissues are good positive controls for CPVL?

CPVL is most abundantly expressed in myeloid cells, particularly monocytes and macrophages. Reliable positive controls include THP-1 cells (especially after PMA differentiation), primary human monocyte-derived macrophages, U937 cells, and dendritic cell preparations. Among tissues, spleen, lung, and placenta lysates give detectable signal. HEK293 and most epithelial lines express CPVL at low or undetectable levels and are useful as low-expression comparators rather than negative controls. For overexpression validation, transient transfection of a full-length CPVL construct in HEK293T provides a clear reference band.

What is a good starting dilution for Western blot with this antibody?

Start at 1:1000 in 5% non-fat milk or BSA in TBST, overnight at 4°C, with 20-30 µg of total lysate per lane. For low-expressing samples, increase loading rather than antibody concentration first. If background is high in macrophage lysates, switch the blocking buffer to 5% BSA and extend washes. The lot has been validated for WB; titrate within a 1:500 to 1:2000 window against your specific lysate to balance signal and background. Use freshly prepared reducing sample buffer because the disulfide-linked processed form can give doublets under partial reduction.

Does this antibody work for IHC and IF, and at what dilution?

The antibody is brand-validated for IHC and immunofluorescence, but lot-specific dilutions for those applications are not certified on this datasheet. For FFPE IHC, heat-induced epitope retrieval in citrate buffer (pH 6.0) is a reasonable first condition, with primary antibody titrated between 1:100 and 1:500. For IF on fixed macrophages, 4% PFA fixation followed by 0.1% Triton X-100 permeabilization preserves the punctate lysosomal staining pattern expected for CPVL. Co-stain with LAMP1 or LAMP2 to confirm lysosomal localization. Titrate against a known positive sample before committing to a large experiment.

Will this antibody cross-react with mouse or rat CPVL?

Reactivity is validated only against human CPVL. Human and mouse CPVL share roughly 80% sequence identity across the mature region, so cross-reactivity with mouse is plausible but has not been confirmed in-house and should be considered predicted at best. If you intend to use the antibody on mouse macrophages or rodent tissue, run a side-by-side blot with a known human-positive control (PMA-differentiated THP-1) and your rodent sample, and confirm the band with a CPVL-knockout or siRNA-depleted control before drawing conclusions.

How should I prepare lysates to preserve CPVL signal?

CPVL is a lysosomal protein, so use a lysis buffer that efficiently solubilizes membrane-bound and luminal lysosomal content. RIPA buffer supplemented with protease inhibitors works well; for native or activity studies use a milder NP-40 or Triton X-100 buffer at pH 7.4. Avoid prolonged incubation at room temperature, since endogenous lysosomal proteases released during lysis can clip CPVL and generate lower molecular weight fragments. Boil samples at 95°C for 5 minutes in reducing Laemmli buffer immediately after lysis. For macrophage samples, briefly sonicate to shear genomic DNA and reduce viscosity.

What controls should I include to confirm band specificity?

Three controls strengthen specificity claims for CPVL. First, an siRNA or shRNA knockdown in THP-1 or primary macrophages should reduce the band at ~54-60 kDa. Second, parallel running of a CPVL overexpression lysate (HEK293T transfected with full-length CPVL) provides a positive reference at the expected size. Third, a low-expressing line such as HeLa or untransfected HEK293 serves as a near-negative comparator. Pre-adsorption with the immunizing antigen, if available, is the cleanest specificity test. Always run a loading control on the same membrane rather than a stripped reblot when quantifying.

How should I store the antibody and how stable is it?

Store the antibody at -20°C upon receipt for long-term storage. After the first thaw, aliquot into low-binding tubes in 10-20 µL volumes to avoid repeated freeze-thaw cycles, which are the most common cause of titre loss for rabbit polyclonals. Working aliquots can be kept at 4°C for up to two weeks if sodium azide is present in the formulation; do not add azide if you plan to use the antibody with HRP-conjugated secondaries in the same incubation. Avoid frost-free freezers, which cycle temperature and degrade antibody activity over time.