HAI-1 (Recombinant)

Frequently Asked Questions

What molecular weight should I expect for recombinant HAI-1 on SDS-PAGE or Western blot?

The HAI-1 recombinant (REC-HAI1) is produced as a soluble ectodomain fragment in HEK293 cells, corresponding to the shed extracellular inhibitory form of the full-length 529-aa protein (UniProt O43278, predicted unglycosylated MW ~58 kDa). Due to N-linked glycosylation added by the HEK293 expression system, the apparent MW on reducing SDS-PAGE typically runs between 65–80 kDa. A diffuse or doublet band in this range is normal and reflects glycan heterogeneity, not degradation. Under non-reducing conditions, disulfide-dependent compaction may shift the apparent MW slightly downward.

Which serine proteases does recombinant HAI-1 inhibit and what are the relevant IC50 values?

REC-HAI1 carries two functional Kunitz inhibitor domains. The first Kunitz domain is the primary inhibitory unit and mediates potent inhibition of matriptase (ST14) and TMPRSS13; reported Ki values for matriptase inhibition are in the low nanomolar range (Ki ~0.3–3 nM depending on the assay format). HAI-1 also inhibits hepatocyte growth factor activator (HGFAC) with similar affinity. The second Kunitz domain contributes structural stability but has weaker intrinsic inhibitory activity. When designing dose-response experiments, titrate REC-HAI1 against your target protease starting at 1–50 nM and adjust based on the protease concentration used.

What activity assay substrate and buffer conditions work best for testing recombinant HAI-1 inhibitory function?

For matriptase inhibition assays, a standard approach is to pre-incubate REC-HAI1 with active matriptase (5–10 nM) for 15–30 min at 37°C in 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.01% Tween-20, then add a fluorogenic substrate such as Boc-Gln-Ala-Arg-AMC or Ac-KQLR-AMC (100 µM) and monitor fluorescence at Ex 380 nm / Em 460 nm. REC-HAI1 is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol — dilute directly into assay buffer; glycerol at ≤1% final concentration does not interfere with fluorogenic readouts.

What starting concentration of recombinant HAI-1 should I use in a matriptase inhibition experiment?

Begin with a molar ratio of REC-HAI1 to matriptase of approximately 5:1 to confirm full inhibition, then perform a serial dilution (e.g., 0.1–100 nM REC-HAI1 against a fixed 2–5 nM matriptase) to determine the IC50 under your specific assay conditions. Enzyme-to-inhibitor ratios, substrate concentration, and ionic strength all shift the apparent IC50, so do not transfer IC50 values between assay formats without re-validation. A no-inhibitor control and a SBTI (soybean trypsin inhibitor) reference standard run in parallel are useful benchmarks.

Can I use REC-HAI1 as a positive control for Western blot with the matched anti-HAI-1 antibody RP-HAI1?

Yes. REC-HAI1 and the matched rabbit polyclonal RP-HAI1 (/anti-hai-1-rabbit-polyclonal-antibody) are validated together for this application. Load 50–200 ng of REC-HAI1 per lane under reducing conditions; the antibody is validated to detect the glycosylated recombinant band at 65–80 kDa. This pairing is particularly useful when blotting conditioned media or cell lysates from epithelial lines known to shed HAI-1 (e.g., LNCaP, Caco-2), where confirming antibody specificity against a defined recombinant standard eliminates ambiguity about band identity.

How much recombinant HAI-1 should I load per lane for Western blot antibody validation with RP-HAI1?

For routine positive control lanes, 100 ng per lane is a reliable starting point when using RP-HAI1 at a 1:1,000–1:2,000 dilution with standard HRP-conjugated secondary antibodies and ECL detection. If signal is weak, increase to 200 ng or reduce antibody dilution to 1:500. On the same blot, include a mammalian cell lysate known to express endogenous HAI-1 (e.g., MCF-7 or Caco-2 total cell lysate, 20–30 µg total protein) to confirm the antibody detects the endogenous form at a comparable apparent MW.

How should I store and handle recombinant HAI-1 to preserve inhibitory activity over time?

REC-HAI1 is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol and should be stored at −20°C in single-use aliquots. Avoid repeated freeze-thaw cycles, which can compromise the disulfide architecture of the Kunitz domains and reduce inhibitory activity. Upon thawing, keep on ice and use within the same working session. Do not dilute into phosphate-buffered saline containing metal chelators (e.g., EDTA) unless required by your assay, as trace metals are not a stability concern for this inhibitor but sudden pH shifts below 6.5 may affect Kunitz domain folding.

Is recombinant HAI-1 glycosylated, and does glycosylation affect its inhibitory activity against matriptase?

REC-HAI1 is expressed in HEK293 cells and carries mammalian N-linked glycosylation, accounting for the ~7–20 kDa shift above the predicted unglycosylated mass. Glycosylation on HAI-1 is primarily found in the stem region outside the Kunitz domains and does not directly contact the protease-binding interface, so inhibitory activity against matriptase and HGFAC is preserved. HEK293 expression was specifically chosen over E. coli or insect cell systems because correct disulfide bond formation within the Kunitz domains — not glycan decoration — is the primary determinant of inhibitor function, and HEK293 cells provide an oxidizing ER environment suited to this requirement.