HTRA-2 (Recombinant)

Frequently Asked Questions

What molecular weight band should I expect for recombinant HTRA2 on SDS-PAGE or Western blot?

Full-length human HTRA2 is 458 amino acids with a predicted unprocessed MW of ~49 kDa. The HEK293-expressed recombinant corresponds to the mature, mitochondria-processed form — the N-terminal mitochondrial targeting sequence (residues ~1–133) is cleaved, yielding the active form at approximately 36 kDa under reducing SDS-PAGE conditions. Glycosylation in the HEK293 system may shift the observed band 1–3 kDa above the theoretical value. Run alongside a pre-stained ladder bracketing 30–50 kDa for reliable sizing.

Which isoform or processing form of HTRA2 is this recombinant — full-length or mature processed?

REC-HTRA2 corresponds to the mature, processed form of HTRA2 (UniProt O43464), beginning after auto-cleavage of the mitochondrial targeting and transmembrane segment. This mirrors the species released into the cytosol during apoptotic stress and retains both the chymotrypsin-like catalytic triad and the C-terminal PDZ domain. It does not include the N-terminal membrane anchor (residues ~1–133), so it is directly relevant to cytosolic apoptosis signalling studies rather than mitochondrial import experiments.

What substrates does HTRA2 cleave and which are recommended for in vitro serine protease activity assays?

HTRA2 (EC 3.4.21.108) exhibits chymotrypsin-like specificity, preferring hydrophobic residues at the P1 position. For in vitro activity assays, β-casein is a commonly used non-specific protein substrate visualised by SDS-PAGE after incubation at 37 °C. For kinetic, fluorimetric readouts, the fluorogenic peptide substrate VDVAD-AMC or Ac-IEPD-AMC can be used (excitation ~360 nm, emission ~460 nm). IAP/BIRC family members — particularly XIAP — serve as physiologically relevant substrates and binding partners for inhibition and competition assays.

What buffer and reaction conditions are recommended for HTRA2 serine protease activity assays?

REC-HTRA2 is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol and is active in this formulation without further exchange. For fluorogenic substrate assays, dilute into assay buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM DTT) to a working concentration of 50–200 nM; include DTT to maintain the catalytic cysteine-adjacent residues in reduced form. Avoid Zn²⁺ or Cu²⁺ ions, which inhibit many serine proteases. Reactions are typically run at 37 °C for 30–60 min. Glycerol from the storage buffer (final <1%) does not measurably affect activity at these dilutions.

What starting concentration of recombinant HTRA2 should I use for an IAP binding or inhibition assay?

For pull-down or co-immunoprecipitation assays with IAP/BIRC family proteins, 0.5–1 µg of REC-HTRA2 per reaction is a reasonable starting point. For enzyme inhibition (IC50) determinations against small-molecule or peptide inhibitors, use 50–100 nM enzyme with substrate at or below Km to maintain linear kinetics. Because HTRA2 forms trimers in solution, ensure equimolar comparisons are based on monomer MW (~36 kDa). Titrate inhibitor across at least eight concentrations spanning three orders of magnitude for reliable curve fitting.

Can I use REC-HTRA2 as a Western blot positive control for the matched RP-HTRA2 antibody?

Yes — this is one of the primary design rationales for pairing REC-HTRA2 with RP-HTRA2. The rabbit polyclonal antibody (SKU: RP-HTRA2; details at /anti-htra-2-rabbit-polyclonal-antibody) was validated against the same recombinant protein. Load 20–50 ng of REC-HTRA2 per lane alongside your cell lysates; the ~36 kDa band provides an unambiguous size reference and confirms antibody performance batch-to-batch. This is particularly useful when optimising lysis conditions for mitochondria-enriched fractions, where endogenous HTRA2 signal can be variable.

How much recombinant HTRA2 should I load for Western blot positive control and what dilution of RP-HTRA2 antibody is recommended?

Load 20–50 ng of REC-HTRA2 per lane on a 12% SDS-PAGE gel; this is sufficient to produce a clean ~36 kDa band without overloading. For RP-HTRA2 (SKU: RP-HTRA2), a primary antibody dilution of 1:500–1:2,000 in 5% non-fat milk/TBST is a reliable starting range based on our in-house validation data. Use HRP-conjugated anti-rabbit secondary at 1:5,000–1:10,000. Chemiluminescent exposure of 30–120 seconds is typically sufficient at these protein loads. Refer to the RP-HTRA2 product page for cell-line-specific validation images.

How should I store and handle REC-HTRA2 to preserve serine protease activity long-term?

REC-HTRA2 is supplied in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol and shipped on dry ice. Store at −20 °C in single-use aliquots immediately upon receipt. Avoid repeated freeze-thaw cycles, which degrade enzyme activity; even two additional cycles can reduce activity by >30% for serine proteases of this class. If using only part of an aliquot, dilute the remainder in the supplied buffer supplemented with 0.05% BSA as a carrier, snap-freeze in liquid nitrogen, and store at −80 °C for short-term use within 2 weeks. Do not store at 4 °C beyond 48 hours.