Anti-Napsin-A Rabbit Polyclonal Antibody

Frequently Asked Questions

What molecular weight should I expect for Napsin-A on a Western blot?

Napsin-A migrates as a mature protein at approximately 33–35 kDa on reducing SDS-PAGE, corresponding to the processed active form. The full-length precursor (420 amino acids, ~47 kDa) is cleaved during maturation, removing the propeptide. In lysates from type II pneumocytes or lung adenocarcinoma cell lines, you will typically observe the 33–35 kDa band. Occasionally, a faint higher-MW species may appear if significant amounts of unprocessed proenzyme are present. Run a positive control (A549 or H1299 lung adenocarcinoma lysate) alongside your samples to confirm band identity.

Which tissues and cell lines serve as good positive controls for Napsin-A?

Napsin-A is highly enriched in normal lung tissue (type II pneumocytes) and renal proximal tubular epithelium. For Western blot, A549, H1299, or NCI-H441 lung adenocarcinoma cell lysates are reliable positive controls. In immunohistochemistry, sections of normal lung parenchyma or clear-cell renal cell carcinoma provide strong cytoplasmic staining. Negative controls include pleural mesothelioma specimens or cell lines such as MSTO-211H, where Napsin-A expression is absent. Avoid squamous-cell carcinoma lines, which are typically Napsin-A-negative and serve as useful negative references in lung cancer studies.

What starting dilution should I use for Western blot and IHC with this antibody?

For Western blot, begin at 1:1000 in 5 percent non-fat milk or BSA in TRIS-buffered saline with Tween-20. This polyclonal antibody was validated at that dilution against human lysates. For immunohistochemistry on formalin-fixed paraffin-embedded sections, a typical range is 1:100 to 1:400; start at 1:200 and titrate based on signal intensity and background in your tissue type. Heat-mediated antigen retrieval in citrate buffer, pH 6.0, is recommended for IHC. Always include a known Napsin-A-positive tissue section in each IHC run to verify staining conditions.

Does this Napsin-A antibody cross-react with mouse or rat samples?

This antibody has been validated against human Napsin-A. Reactivity with mouse or rat homologues is predicted but not experimentally confirmed. Human and mouse Napsin-A share approximately 75 percent sequence identity, so cross-reactivity is plausible. If you plan to work with rodent lung or kidney lysates, perform an initial titration experiment with positive-control mouse lung tissue alongside a human sample. Compare band patterns and molecular weights. If a specific band at 33–35 kDa appears, validation is promising; absence of signal or additional non-specific bands warrants caution. Contact us if you require validated cross-species data.

Why do I see high background in IHC of lung tissue with Napsin-A antibody?

High background in lung IHC is often due to endogenous biotin in type II pneumocytes or inadequate blocking. Use a biotin-free detection system (polymer-HRP or fluorophore-conjugated secondary) if employing avidin-biotin amplification. Ensure sufficient blocking with normal serum matching your secondary antibody host species, typically 10 percent for 30 minutes. Protease-rich lung homogenates can also cause non-specific binding; avoid over-digestion during antigen retrieval. Finally, polyclonal antibodies may require slightly higher blocking stringency than monoclonals. Include a no-primary-antibody control slide to distinguish true signal from detection-system artifacts.

Can Napsin-A antibody distinguish lung adenocarcinoma from squamous-cell carcinoma?

Yes, Napsin-A is one of the most reliable markers for this distinction in diagnostic pathology. Lung adenocarcinomas express Napsin-A in 70–85 percent of cases, reflecting their derivation from distal airway or type II pneumocyte lineages. Squamous-cell carcinomas are almost uniformly Napsin-A-negative. In a research or diagnostic panel, combine Napsin-A with TTF-1 for adenocarcinoma and p40 or p63 for squamous differentiation. Napsin-A also helps exclude metastatic carcinomas and mesotheliomas, which lack expression. Interpret staining in the context of morphology and additional lineage markers for robust classification.

How should I store this Napsin-A antibody, and what is the expected shelf life?

Store the antibody at –20 °C in single-use aliquots to avoid repeated freeze-thaw cycles, which can reduce titre and increase aggregation. Do not store diluted antibody; prepare working dilutions fresh for each experiment. The product is supplied in a glycerol-containing buffer that remains stable for at least 12 months at –20 °C from the date of receipt. For long-term storage beyond one year, –80 °C is preferable. Avoid prolonged exposure to room temperature. If you observe precipitate after thawing, centrifuge briefly at 10,000 × g before use. Check lot-specific expiry on the datasheet.

What sample preparation steps optimize Napsin-A detection in Western blot?

Napsin-A is a secreted protein, so both cell lysates and culture supernatants are relevant sample types. For adherent cells, harvest in RIPA or NP-40 lysis buffer supplemented with protease inhibitors (leupeptin, pepstatin A) to prevent auto-proteolysis. Load 20–30 µg total protein per lane. For secreted Napsin-A in conditioned medium, concentrate by TCA precipitation or ultrafiltration (10-kDa cutoff) and load equivalent volumes. Use reducing conditions (DTT or β-mercaptoethanol) to fully denature the protein. Transfer to PVDF rather than nitrocellulose for sharper bands in the 30–40 kDa range. A positive-control lung lysate run in parallel ensures antibody performance.