HTRA-4 (Recombinant)

Frequently Asked Questions

What is the expected molecular weight of recombinant HTRA-4 on SDS-PAGE and Western blot?

Full-length human HTRA4 (476 aa, UniProt P83105) has a predicted unprocessed MW of ~52 kDa. Because this recombinant is expressed in HEK293 cells, the N-terminal signal peptide is cleaved co-translationally, which typically shifts the apparent MW to ~49–50 kDa under reducing SDS-PAGE. Glycosylation from the mammalian expression system may cause the band to run slightly higher or as a diffuse smear — commonly observed between 50–60 kDa. We recommend running a broad-range ladder (10–250 kDa) and not relying solely on predicted MW when interpreting gels.

Is recombinant HTRA-4 produced as a full-length protein or a processed/truncated form?

The HEK293-expressed recombinant corresponds to the mature secreted form of HTRA4 following signal-peptide cleavage of the N-terminal leader sequence. It retains the intact trypsin-like serine protease catalytic domain and the C-terminal PDZ regulatory domain — the two structural elements essential for activity and substrate gating. No artificial truncations are introduced. This is particularly relevant for researchers studying PDZ-mediated regulation or oligomerization, as both domains are preserved in the correct eukaryotic folding context.

What substrates can I use to measure HTRA-4 serine protease activity in vitro?

HTRA4 is a trypsin-like serine protease; fluorogenic peptide substrates commonly used for HtrA-family members include Boc-Ala-Pro-Arg-AMC and H-Ala-Ala-Pro-Phe-AMC (the latter used for HTRA1/2 benchmarking). A casein-based degradation assay (fluorescein-labeled casein, e.g., EnzChek) also provides a straightforward endpoint readout. Start with 50–200 nM recombinant HTRA4 in assay buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl) at 37°C; substrate concentrations of 100–500 µM are typical. Confirm activity is serine-protease-dependent by including 1 mM PMSF as a negative control.

What buffer conditions are optimal for HTRA-4 protease activity assays?

The storage buffer — 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol — is compatible with direct use in activity assays; simply dilute to your desired enzyme concentration. Glycerol above 1% can quench some fluorogenic AMC substrates, so dilute at least 10-fold into assay buffer before adding substrate. HTRA4 serine protease activity is optimal near pH 7.0–8.0; avoid acidic conditions below pH 6.5, which suppress the catalytic Ser-His-Asp triad. Do not include EDTA, as divalent ions are not required but chelators may interfere with buffer stability at higher concentrations.

What starting concentration of HTRA-4 recombinant protein should I use for an IC50 inhibitor screening assay?

For inhibitor IC50 determinations, begin titrations with 50–100 nM HTRA4 in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, assayed with a fluorogenic substrate at a concentration near its apparent Km (empirically determined, but 200 µM is a reasonable starting point for AMC-based peptides). Running enzyme at a concentration well below inhibitor concentrations avoids tight-binding artifacts. Include PMSF (1 mM) as a serine protease positive-control inhibitor to validate assay window before committing inhibitor libraries. A Z′ factor ≥ 0.5 should be confirmed before full-plate screening.

Can I use recombinant HTRA-4 as a Western blot positive control for the RP-HTRA4 rabbit polyclonal antibody?

Yes — this is one of the primary intended uses of REC-HTRA4. The matched antibody, RP-HTRA4 (see /anti-htra-4-rabbit-polyclonal-antibody), is produced and validated in the same laboratory against HTRA4 antigen, guaranteeing epitope compatibility. Load 20–50 ng of REC-HTRA4 per lane alongside your cell lysate samples; this amount reliably produces a distinct band at ~50–55 kDa under standard reducing conditions with most ECL detection systems. This lane serves as a size reference and antibody sensitivity control simultaneously, particularly useful when validating RP-HTRA4 on a new lysate background or tissue type.

How much recombinant HTRA-4 should I load for a Western blot positive control, and at what antibody dilution?

Load 20–50 ng of REC-HTRA4 per lane for a strong, clean positive control band. At this loading, RP-HTRA4 (rabbit polyclonal, SKU: RP-HTRA4) typically performs well at a primary antibody dilution of 1:500–1:2,000 in 5% non-fat dry milk/TBST with 1-hour room temperature incubation, though optimal dilution should be confirmed on your detection platform. Overloading beyond 100 ng can produce a smeared band due to the glycosylated nature of the HEK293-expressed protein. Always run a no-primary-antibody lane in parallel to confirm signal specificity.

How should I handle, dilute, and store recombinant HTRA-4 to preserve activity over time?

REC-HTRA4 is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol and is stable at −20°C for at least 12 months from the date of manufacture when stored in single-use aliquots. Repeated freeze-thaw cycles measurably reduce serine protease activity — plan aliquot sizes around your typical single-experiment usage. For dilutions, use the storage buffer or your assay buffer supplemented with 0.1% BSA as a carrier to prevent loss of enzyme to tube walls at low concentrations (below ~10 nM). Avoid vortexing; mix gently by pipetting. Do not store at 4°C for extended periods.