SLPI (Recombinant)

Frequently Asked Questions

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

The mature SLPI sequence spans 132 amino acids, giving a predicted MW of ~11.7 kDa. On reducing SDS-PAGE this recombinant typically resolves as a diffuse band around 12–14 kDa; the slight upward shift reflects HEK293-derived N-linked glycosylation. Under non-reducing conditions, migration can appear slightly different due to the eight intact disulfide bonds within the two WAP domains. When using this protein as a positive control on Western blot with RP-SLPI, load 20–50 ng per lane for a clean, well-defined signal.

Is this recombinant SLPI the full-length mature form or does it include the signal peptide?

This preparation corresponds to the mature secreted form of human SLPI (UniProt P03973), beginning after cleavage of the 23-amino-acid signal peptide — residues 24–157 of the full precursor, yielding the 132-residue functional inhibitor. Both WAP domains (N-terminal and C-terminal) are present and correctly folded. The C-terminal WAP domain carries the primary serine protease-inhibitory reactive site loop; the HEK293 expression system supports the full complement of eight disulfide bonds required for this native architecture.

What proteases does recombinant SLPI inhibit and what substrates are used to measure its activity?

SLPI is a tight-binding inhibitor of neutrophil elastase, cathepsin G, trypsin, and chymotrypsin. For kinetic inhibition assays, human neutrophil elastase (HNE) is the most commonly used target; the standard fluorogenic substrate is MeO-Suc-Ala-Ala-Pro-Val-AMC (200–400 µM). Cathepsin G activity is typically monitored with Suc-Ala-Ala-Pro-Phe-AMC. Assays are run in 50 mM HEPES pH 7.4, 150 mM NaCl, 0.01% Tween-20 at 37°C. Pre-incubate SLPI with the target protease for 15–30 minutes before adding substrate to allow equilibrium binding.

What is the IC50 or Ki of recombinant SLPI against human neutrophil elastase, and what starting concentration should I use?

SLPI is a slow, tight-binding inhibitor of HNE with a Ki in the low nanomolar range (reported values typically 0.5–5 nM depending on assay format and enzyme source). For IC50 determination, titrate SLPI from 0.1 nM to 100 nM against a fixed HNE concentration (1–5 nM). A practical starting concentration for inhibition profiling is 10–50 nM recombinant SLPI. Because this is a slow-binding inhibitor, apparent IC50 values are incubation-time-dependent; use progress-curve analysis or Morrison's tight-binding equation for accurate Ki extraction.

What buffer conditions are compatible with recombinant SLPI activity assays, and does the storage buffer interfere?

This recombinant is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol. For activity assays, dilute at least 1:10 into assay buffer (e.g., 50 mM HEPES pH 7.4, 150 mM NaCl, 0.01% Tween-20) to reduce glycerol to ≤1%, which does not measurably affect HNE or cathepsin G kinetics. SLPI is notably acid-stable; it retains inhibitory activity down to pH 5, making it compatible with cathepsin-G assays run at mildly acidic pH. Avoid reducing agents such as DTT or TCEP — these will disrupt the disulfide scaffold and abolish activity.

Can I use recombinant SLPI as a positive control for Western blot with your matched anti-SLPI antibody (RP-SLPI)?

Yes — this is the primary intended pairing. RP-SLPI (/anti-slpi-rabbit-polyclonal-antibody) was validated directly against this HEK293-expressed recombinant. Load 20–50 ng of recombinant SLPI per lane under reducing conditions; expect a band at 12–14 kDa. Use RP-SLPI at 1:500–1:2,000 dilution with standard HRP-conjugated secondary. This positive control is especially useful when detecting endogenous SLPI in bronchoalveolar lavage, saliva, or seminal plasma samples, where band position and intensity can be confirmed against the recombinant standard.

How much recombinant SLPI should I load for Western blot positive control when validating RP-SLPI antibody?

For antibody validation purposes with RP-SLPI, 20 ng is generally sufficient to produce a strong signal at 12–14 kDa; 50 ng gives a robust band even at higher antibody dilutions (up to 1:5,000). If you are running a dilution series to determine the linear detection range of RP-SLPI, a 5-point series from 5 ng to 100 ng works well. Load alongside a cell lysate positive control (e.g., A549 or Calu-3, which express endogenous SLPI) to confirm antibody specificity in a complex matrix simultaneously.

How should I handle, dilute, and store recombinant SLPI to preserve activity over time?

Upon receipt, briefly centrifuge the vial and store at -20°C in single-use aliquots — repeated freeze-thaw cycles progressively reduce inhibitory activity due to disulfide scrambling. For working dilutions, use assay-compatible buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 0.01% BSA) rather than PBS alone; BSA at 0.01–0.1% prevents adsorptive losses at low concentrations (≤10 nM). Diluted working stocks should be used within 24 hours and kept on ice. When stored correctly, activity is stable for at least 12 months from date of receipt. Purity is >95% by SDS-PAGE; endotoxin is <0.1 EU/µg by LAL.