PRSS-16 (Recombinant)

Frequently Asked Questions

What molecular weight should I expect for recombinant PRSS-16 on SDS-PAGE or Western blot?

The unmodified 514-amino-acid sequence predicts a molecular weight of approximately 57 kDa. However, because REC-PRSS16 is expressed in HEK293 cells, N-linked glycosylation is expected to shift the apparent MW on SDS-PAGE to roughly 60–70 kDa, depending on gel conditions and glycan heterogeneity. Under reducing conditions, a single predominant band in this range confirms intact, properly folded protein. If you observe a doublet, this likely reflects differential glycosylation rather than contamination — purity is confirmed >90% by SDS-PAGE on every lot.

Is recombinant PRSS-16 full-length or a processed/truncated form, and does it contain the signal peptide?

The construct is based on the full 514-residue coding sequence of PRSS16 (UniProt Q9NQE7). Like other trypsin-family serine proteases, PRSS16 is likely produced as a zymogen requiring proteolytic activation; the recombinant is expressed in HEK293 cells and supplied in active form, but researchers investigating zymogen processing or propeptide cleavage should verify the exact expressed boundaries in the certificate of analysis for their specific lot. Signal peptide removal by the HEK293 secretory machinery is anticipated.

What substrates does PRSS-16 cleave and which fluorogenic peptide substrates work for activity assays?

PRSS16 is a serine endopeptidase of the trypsin family, so it preferentially cleaves after basic residues (Arg, Lys). Fluorogenic substrates such as Boc-Gln-Ala-Arg-AMC or Z-Arg-Arg-AMC are reasonable starting points for activity assays. Measure fluorescence at Ex/Em 380/460 nm. Because PRSS16's precise in-vivo cleavage specificity in antigen-processing pathways is not fully resolved, we recommend running a substrate panel alongside a trypsin control to establish baseline hydrolysis rates and validate that your preparation is active prior to inhibitor studies.

What buffer conditions and pH are optimal for PRSS-16 serine protease activity assays?

REC-PRSS16 is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol — conditions compatible with direct dilution into most activity assay buffers. For enzymatic assays, a working buffer of 50 mM Tris-HCl or HEPES pH 7.5–8.0, 100–150 mM NaCl, 0.01% Triton X-100 (to reduce surface adsorption) is a practical starting point. Serine proteases in this family are typically inhibited by PMSF or AEBSF — avoid these in assay buffers. Pre-warm enzyme to 25°C or 37°C for 10 minutes before initiating reactions.

What starting concentration of recombinant PRSS-16 should I use for an IC50 inhibitor assay?

For IC50 determinations, a typical starting point is 5–20 nM REC-PRSS16 in the assay well, depending on your substrate concentration and the Km of the substrate chosen. Titrate enzyme concentration first to confirm linear substrate hydrolysis over the assay time window (30–60 min is typical). Keep enzyme below 10% substrate conversion at the chosen time point to maintain initial-rate conditions. Dilute from the stock in assay buffer supplemented with 0.1% BSA to minimize adsorption losses, particularly at sub-nanomolar enzyme concentrations.

Can I use REC-PRSS16 as a positive control for Western blot with the matched PRSS-16 antibody (RP-PRSS16)?

Yes — REC-PRSS16 and the matched rabbit polyclonal RP-PRSS16 (see /anti-prss-16-rabbit-polyclonal-antibody) are produced and validated together in the same lab, making this the most reliable positive-control pairing available. The antibody is validated for Western blot and is guaranteed compatible with this recombinant. Load 10–50 ng of REC-PRSS16 per lane under reducing conditions; expect a band at ~60–70 kDa. This loading range typically yields a clean, strong signal without saturation, allowing you to co-run it alongside cell lysate lanes for direct band-size confirmation.

How much recombinant PRSS-16 should I load as a Western blot positive control, and what band should I see?

Load 20–50 ng of REC-PRSS16 per lane on a 10–12% SDS-PAGE gel under reducing conditions. Paired with RP-PRSS16 (SKU: RP-PRSS16), expect a primary band at approximately 60–70 kDa, reflecting the glycosylated HEK293-expressed form. This range consistently produces a detectable band without overwhelming nearby lanes in multiplexed blots. If you are optimizing RP-PRSS16 antibody dilution simultaneously, running a 2-fold dilution series of the recombinant (10–100 ng) alongside your lysate samples is an efficient way to establish both antibody sensitivity and linearity in a single experiment.

How should I store and handle recombinant PRSS-16 to maintain activity, and what is the shelf life?

REC-PRSS16 is shipped on dry ice and should be stored at -20°C immediately upon receipt in the single-use aliquots provided. Repeated freeze-thaw cycles measurably reduce serine protease activity — aliquot further if your working volume is smaller than the unit size. In storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol), the protein is stable for at least 12 months at -20°C when handled correctly. Once thawed, keep on ice and use within the same working session. Adding 0.1% BSA as a carrier can help maintain activity during prolonged dilution steps at low protein concentrations.