Napsin (Recombinant)

Frequently Asked Questions

What molecular weight band should I expect for recombinant Napsin-A on SDS-PAGE or Western blot?

The full-length Napsin-A sequence (420 residues, UniProt O96009) predicts a polypeptide backbone of approximately 45 kDa, but because this recombinant is produced in HEK293 cells it carries mammalian N-linked glycosylation. On reducing SDS-PAGE you should routinely observe a diffuse band in the 50–55 kDa range, slightly higher than the calculated mass. Treating the sample with PNGase F prior to electrophoresis collapses this to the ~45 kDa backbone and is a useful confirmation of protein identity.

Is this recombinant Napsin the mature processed form or does it include the propeptide?

The construct spans the full 420-residue sequence, which includes the N-terminal signal peptide and propeptide region characteristic of pepsin-family aspartyl proteases. In HEK293 expression the signal peptide is cleaved co-translationally during secretion, but propeptide processing to the fully mature form may be partial. If your assay requires strictly propeptide-free enzyme, incubation at pH 3.5–4.0 for 30 minutes at 37 °C can promote autocatalytic propeptide removal, consistent with the acid-activation mechanism described for pepsin-family members.

What substrates and assay conditions work best for measuring recombinant Napsin-A protease activity?

Napsin-A is an aspartyl protease most active at acidic pH. A well-characterized fluorogenic substrate is the FRET pair MCA-Lys-Pro-Ala-Glu-Phe-Phe-Ala-Leu-Lys(DNP) (or similar pepsin-type substrates), assayed in 50 mM sodium acetate, 150 mM NaCl, pH 4.0–4.5, at 37 °C. Excitation/emission at 320/405 nm is standard for MCA-based substrates. For a starting point, titrate enzyme at 5–50 nM against 10–100 µM substrate and determine Km and Vmax empirically, as specific activity varies with propeptide processing state of the preparation.

What is a good starting concentration of recombinant Napsin-A for protease activity assays?

For initial activity screening, 10–25 nM recombinant Napsin-A in the assay volume is a practical starting range. Dilute from the stock into the assay buffer (50 mM sodium acetate pH 4.0, 150 mM NaCl) immediately before use, not into the 50 mM Tris-HCl pH 7.5 storage buffer, since enzyme is most active and stable under acidic assay conditions during the assay window. Include a no-enzyme blank and a pepstatin A control (1 µM) to confirm aspartyl protease-dependent signal and to rule out contaminating protease activity.

Can I use recombinant Napsin-A as a positive control antigen with the RP-Napsin rabbit polyclonal antibody on Western blot?

Yes — this is the primary validated pairing. The matched antibody RP-Napsin (/anti-napsin-rabbit-polyclonal-antibody) was raised and validated in the same laboratory against Napsin-A antigen. Load 20–50 ng of recombinant Napsin-A per lane on a 10–12% SDS-PAGE gel. After transfer, probe with RP-Napsin at the manufacturer's recommended dilution (typically 1:500–1:2,000 in 5% non-fat milk/TBST). You should detect a clean band at 50–55 kDa, confirming antibody specificity before moving to cell lysate or tissue samples.

How much recombinant Napsin-A should I load as a Western blot positive control alongside lung tissue lysate?

For a side-by-side positive control lane, 20–50 ng of recombinant Napsin-A is typically sufficient when using RP-Napsin at standard working dilutions. Load recombinant protein in the same SDS-PAGE sample buffer (with β-mercaptoethanol or DTT) as your lysate lanes to keep running conditions identical. Because the recombinant band (50–55 kDa, glycosylated) may migrate slightly differently from endogenous Napsin in some lysates depending on glycoform heterogeneity, run both to establish the expected band position for your specific antibody lot and detection system.

How should I store and handle recombinant Napsin-A to preserve activity over multiple experiments?

Store at -20 °C in single-use aliquots immediately upon receipt. The supplied buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol — is formulated to maintain enzyme integrity during frozen storage. Repeated freeze-thaw cycles measurably reduce activity and should be avoided; aliquot at 10–20 µg per tube during initial receipt if your working quantities are small. Once thawed, hold on ice and use within the same working session. Do not dilute working stocks in pH 4.0 assay buffers until immediately before the assay, and do not re-freeze diluted material.

What endotoxin level and purity can I expect in the recombinant Napsin-A preparation?

Purity is >90–95% by reducing SDS-PAGE with Coomassie staining, verified for each lot. Endotoxin is <0.1 EU/µg as determined by the LAL (Limulus Amebocyte Lysate) chromogenic assay. These specifications matter particularly for cell-based assays — Napsin-A is expressed in alveolar type II cells, so researchers using this recombinant in pneumocyte culture models need confidence that observed cytokine responses or proliferative effects are not LPS artifacts. A certificate of analysis with lot-specific SDS-PAGE and endotoxin data is available on request.