HTRA-1 (Recombinant)

Frequently Asked Questions

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

The full-length HTRA1 precursor (UniProt Q92743) has a predicted MW of ~51 kDa, but the HEK293-expressed recombinant typically migrates at 55–60 kDa under reducing SDS-PAGE conditions due to N-linked glycosylation. Under non-reducing conditions, trimeric forms may be partially visible as higher-MW species. If you are running a Western blot positive control alongside the matched rabbit polyclonal antibody (SKU: RP-HTRA1), expect the primary band at ~57 kDa. Load 50–100 ng per lane for a clean, unambiguous signal without saturation.

Is this recombinant HTRA-1 the full-length protein or the processed mature protease form?

The recombinant corresponds to the secreted mature form of HTRA1 — the signal peptide (residues 1–22) and IGFBP domain are removed during processing, leaving the trypsin-like serine protease domain and C-terminal PDZ domain intact. This is the biologically relevant extracellular species and the form used in TGF-β suppression and ECM substrate cleavage studies. The catalytic triad (His220, Asp250, Ser328 in canonical numbering) is intact and the enzyme is supplied in its active trimeric configuration, as confirmed by activity assay at time of QC.

What substrates and assay conditions work best for measuring recombinant HTRA-1 protease activity in vitro?

HTRA1 cleaves the fluorogenic peptide substrate VPR-AMC (Val-Pro-Arg-7-amino-4-methylcoumarin) efficiently in activity assays. A practical starting setup: 50 nM recombinant HTRA1 in assay buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM DTT), 100 µM VPR-AMC, 37 °C, read at Ex/Em 360/460 nm. For ECM substrate assays (e.g., fibronectin, decorin, or aggrecan fragments), incubate 0.5–2 µg recombinant HTRA1 with 5–10 µg substrate at 37 °C for 2–16 hours and resolve by SDS-PAGE. DTT is important — HTRA1 activity is sensitive to oxidizing conditions.

What starting concentration of recombinant HTRA-1 should I use for TGF-β pathway suppression experiments in cell culture?

Published cell-based studies typically use 0.1–1 µg/mL recombinant HTRA1 added to conditioned medium or directly to culture medium to observe suppression of TGF-β family signalling via cleavage of extracellular ligand-binding complexes. A dose-response from 50 ng/mL to 2 µg/mL is a reasonable titration range for your first experiment. Confirm activity retention before adding to cells using the VPR-AMC fluorogenic assay; inactive or partially degraded preparations will give ambiguous pathway results. The storage buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol) is compatible with dilution into serum-containing media at these concentrations.

How does the PDZ domain affect HTRA-1 activity and should I account for it when designing inhibitor IC50 assays?

HTRA1's C-terminal PDZ domain acts as an autoinhibitory regulator that gates substrate access to the catalytic cleft. In solution at low concentrations, PDZ-mediated autoinhibition can reduce apparent Vmax, which means IC50 values for competitive inhibitors (e.g., peptidomimetic or small-molecule serine protease inhibitors) are sensitive to enzyme concentration. For reproducible IC50 determinations, fix enzyme concentration at 25–50 nM and pre-incubate inhibitor with enzyme for 15–30 minutes at 37 °C before substrate addition. Displacement of PDZ-mediated inhibition by allosteric activators (e.g., unfolded protein substrates) can shift the apparent IC50, so note the activation state of your enzyme in the methods.

Can I use recombinant HTRA-1 as a positive control for Western blot with the matched RP-HTRA1 rabbit polyclonal antibody?

Yes — this is a primary validated use case for pairing REC-HTRA1 with the matched rabbit polyclonal antibody (SKU: RP-HTRA1). Load 50–100 ng of recombinant HTRA1 alongside your cell lysate or tissue samples. The antibody detects the ~57 kDa glycosylated recombinant band cleanly under standard reducing conditions. Because both products are produced and validated in the same laboratory, lot-to-lot consistency between the recombinant and antibody is tightly controlled. RP-HTRA1 is validated for Western blot; cross-reactivity with mouse HTRA1 is predicted based on sequence homology but should be confirmed empirically in your model system.

How much recombinant HTRA-1 should I load for Western blot when validating the RP-HTRA1 antibody against tissue lysates?

For antibody validation side-by-sides, load 50 ng of REC-HTRA1 as the positive control lane adjacent to your tissue or cell lysates. At this amount, RP-HTRA1 at a 1:1,000–1:2,000 primary antibody dilution gives a prominent band at ~57 kDa after standard ECL detection with a 1–5 minute exposure. Loading above 200 ng risks saturation artifacts and diffuse banding from the glycan heterogeneity. If signal is weak in your lysate lanes, rule out antigen loss from sample boiling in non-reducing buffer — HTRA1 epitopes in RP-HTRA1's target region (residues 46–460) are best preserved under standard reducing Laemmli conditions.

How should I store and handle recombinant HTRA-1 to maintain protease activity over time?

Store at -20 °C in single-use aliquots. The supplied buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol) stabilizes the trimeric enzyme during freeze-thaw, but repeated freeze-thaw cycles progressively reduce specific activity — plan aliquot sizes accordingly (5–10 µg per tube works well for most users). On ice during experiments, HTRA1 is stable for at least 8 hours. Avoid diluting into buffers lacking glycerol for long-term storage; if diluting for cell-based work, use carrier protein (0.1% BSA) for concentrations below 10 µg/mL. Verify activity by VPR-AMC assay from a fresh aliquot before critical experiments.