Anti-Napsin-A Rabbit Polyclonal Antibody

Frequently Asked Questions

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

Napsin-A migrates at approximately 42–45 kDa on reduced SDS-PAGE, corresponding to the mature processed form. The full-length precursor (pre-pro-enzyme) is 420 amino acids with a predicted mass near 46 kDa, but the signal peptide and activation peptide are cleaved during maturation and secretion. You may occasionally observe higher molecular weight species around 50–55 kDa representing glycosylated or incompletely processed forms, particularly in cell lysates versus conditioned media. The mature secreted protease is the predominant form in lung tissue and most adenocarcinoma samples.

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

We recommend starting at 1:1000 dilution for Western blot applications with this rabbit polyclonal antibody, as validated in our hands. This dilution typically provides robust signal from lung adenocarcinoma cell lysates or tissue homogenates when loading 20–30 µg total protein per lane. If your samples express Napsin-A at lower levels or you observe high background, you may need to optimize between 1:500 and 1:2000. Always include a positive control lysate from A549 or H1299 lung adenocarcinoma cells to confirm antibody performance before troubleshooting sample-specific issues.

Does this Napsin antibody distinguish between Napsin-A and Napsin-B isoforms?

This antibody is specific for Napsin-A (gene name NAPSA, UniProt O96009) and does not cross-react with Napsin-B (encoded by the NAPSB gene). Napsin-B shares only limited sequence homology with Napsin-A and exhibits different tissue distribution, primarily in kidney and testis rather than lung. The immunogen used to generate this polyclonal antibody corresponds to regions unique to the Napsin-A protein sequence. If your research requires detection of Napsin-B, you will need a separate antibody designed against that specific isoform.

Will this rabbit polyclonal Napsin antibody work for immunohistochemistry on FFPE lung tissue?

Yes, This antibody is validated for Western blot; immunohistochemistry/immunofluorescence validation is in progress applications on formalin-fixed paraffin-embedded tissue. Napsin-A shows characteristic cytoplasmic granular staining in type II pneumocytes of normal lung and diffuse cytoplasmic positivity in approximately 80% of pulmonary adenocarcinomas. Antigen retrieval is typically required; heat-mediated retrieval in citrate buffer pH 6.0 is a reliable starting point. Optimal dilution for IHC may differ from Western blot and should be titrated on your specific tissue sections, but 1:100 to 1:500 is a reasonable starting range.

Is this Napsin-A antibody validated in species other than human?

This antibody is validated specifically for human Napsin-A. Cross-reactivity with mouse or rat Napsin-A is not confirmed and should be considered predicted rather than demonstrated. Human and mouse Napsin-A share approximately 75–80% amino acid identity, so cross-reactivity is plausible, but you should verify signal specificity using knockout tissue or cells if working with rodent samples. If your experimental model requires robust detection in mouse lung, we recommend validating this antibody in parallel with a known positive control and considering a species-specific antibody if signal is weak or equivocal.

What positive and negative control samples should I use when validating this Napsin antibody?

For Western blot, A549 or H1299 lung adenocarcinoma cell lysates serve as strong positive controls, as both cell lines express endogenous Napsin-A. Normal adult lung tissue homogenate also works well. HeLa or HEK293 cells are useful negative controls since they lack detectable Napsin-A expression. For IHC, normal lung parenchyma (type II pneumocytes positive) and lung adenocarcinoma tissue are ideal positives, while squamous cell carcinoma or small cell lung cancer typically show minimal to absent staining. Renal proximal tubules also express Napsin-A and can serve as an additional positive tissue control.

How should I store this Napsin 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 antibody activity over time. The 100 µg vial is provided as a liquid or lyophilized preparation; verify format on your product datasheet. If liquid, supplement with carrier protein such as 0.1% BSA or 50% glycerol to enhance stability during storage. Properly stored aliquots typically remain stable for at least 12 months at –20°C. Avoid prolonged storage at 4°C for working aliquots; prepare only what you need for one week of experiments.

Why do I see multiple bands on my Napsin-A Western blot?

Multiple bands likely reflect differential glycosylation or proteolytic processing of Napsin-A. The mature enzyme migrates at 42–45 kDa, but you may detect a minor band at 50–55 kDa representing the glycosylated precursor or pro-form. Lower molecular weight fragments below 40 kDa can arise from autocatalytic cleavage, especially in lysates stored without protease inhibitors, since Napsin-A is an active aspartyl protease. Additionally, some lung adenocarcinoma cells produce alternatively processed forms. Include fresh protease inhibitors during lysis and compare your pattern to a validated positive control to distinguish biological complexity from degradation artifacts.