Recombinant Human HTRA2 Protein: Handling & Usage Protocol

Recombinant Human HTRA2 Protein is a purified mitochondrial serine protease used in apoptosis research, biochemical assays, and mechanistic studies of the intrinsic cell-death pathway.

Product Name

Recombinant Human HTRA2 Protein

SKU

rp365977

Protein Class

Serine protease / PDZ domain-containing mitochondrial protein

Biological Function

Promotes apoptosis by cleaving IAP family proteins and activating caspase cascades upon mitochondrial release

Primary Applications

Apoptosis assays, serine protease activity assays, protein interaction studies, western blot positive control

Recommended Storage

Store lyophilized protein at -20°C to -80°C; avoid repeated freeze-thaw cycles

Reference Number

absea#PP-1616

Background and Biological Significance of HTRA2

HTRA2 (High-Temperature Requirement Serine Protease A2), also known as Omi, is a nuclear-encoded mitochondrial serine protease that plays a pivotal role in regulating apoptosis and mitochondrial protein quality control. Under normal physiological conditions, HTRA2 resides in the intermembrane space of mitochondria, where its PDZ domain facilitates protein–protein interactions critical for maintaining mitochondrial homeostasis.

Upon induction of apoptotic stimuli, HTRA2 is released from mitochondria into the cytosol. Once released, it promotes programmed cell death through two complementary mechanisms: (1) direct serine protease activity that degrades inhibitor of apoptosis proteins (IAPs) such as XIAP, and (2) IAP-binding that relieves caspase inhibition, collectively amplifying the caspase cascade. Loss-of-function mutations in HTRA2 have been associated with neurodegeneration, including Parkinson's disease-like phenotypes in animal models, underscoring its translational relevance.

Recombinant Human HTRA2 Protein enables researchers to dissect these pathways in cell-free systems, validate antibody reagents, and establish quantitative protease activity assays, making it an indispensable tool for apoptosis biology laboratories.

Materials and Reagents Required

Before beginning the protocol, ensure the following materials are available:

• Recombinant Human HTRA2 Protein (Aladdin Scientific, SKU: rp365977)
• Sterile, nuclease-free deionized water or reconstitution buffer as specified on the Certificate of Analysis (CoA)
• Phosphate-buffered saline (PBS), pH 7.4, optionally supplemented with 0.1% BSA as a carrier protein to minimize surface adsorption losses
• Low-binding microcentrifuge tubes (1.5 mL)
• Calibrated micropipettes and sterile filter tips
• Dry ice or liquid nitrogen for snap-freezing aliquots
• Appropriate assay buffers depending on downstream application (e.g., serine protease activity buffer, SDS-PAGE sample buffer for western blot)
• Protease inhibitor cocktail (if preparing cell lysates for co-incubation controls)
• Personal protective equipment (PPE): lab coat, nitrile gloves, eye protection

Consult the product-specific Certificate of Analysis supplied with each lot for confirmed protein concentration, purity, and recommended buffer composition prior to use.

Step-by-Step Reconstitution and Handling Protocol

Step 1 – Equilibration. Remove the vial from -80°C (or -20°C) storage and allow it to equilibrate at room temperature for 10–15 minutes before opening. Do not use a heat block or water bath, as localized heating can denature the protein.

Step 2 – Reconstitution. Briefly centrifuge the vial (1,000 × g, 10 seconds) to collect any lyophilized material from the cap and walls. Add the volume of sterile reconstitution buffer specified on the CoA slowly along the inner wall of the vial. Gently swirl—do not vortex—and allow the protein to dissolve for 10 minutes at room temperature. Vigorous mixing can introduce air bubbles and promote aggregation.

Step 3 – Verification of dissolution. Visually inspect the solution for clarity. Slight opalescence may be acceptable; visible aggregates or precipitates indicate suboptimal reconstitution. If aggregates persist, centrifuge at 10,000 × g for 5 minutes at 4°C and use the supernatant.

Step 4 – Aliquoting. Divide the reconstituted protein into single-use aliquots in low-binding microcentrifuge tubes to avoid repeated freeze-thaw cycles, which degrade protease activity. Snap-freeze aliquots on dry ice or in liquid nitrogen before transferring to -80°C for long-term storage.

Step 5 – Working dilutions. Prepare working dilutions fresh on the day of each experiment using the recommended assay buffer. Include 0.1% BSA in the diluent when working at very low protein concentrations (<10 µg/mL) to prevent adsorption to tube surfaces.

Step 6 – Activity confirmation (recommended). For protease activity assays, confirm HTRA2 serine protease activity using a fluorogenic peptide substrate (e.g., casein-FITC or a custom PDZ-domain substrate) before proceeding to primary experiments. Record specific activity relative to protein concentration for lot-to-lot comparisons.

Downstream Applications and Experimental Considerations

Serine Protease Activity Assays: HTRA2 exhibits trypsin-like serine protease activity and can be assayed using synthetic fluorogenic substrates. Assay conditions should be optimized for pH (typically 7.4–8.0), temperature (37°C), and ionic strength. Include appropriate serine protease inhibitors (e.g., PMSF or 4-(2-aminoethyl)benzenesulfonyl fluoride) as negative controls to confirm on-target activity.

Apoptosis Pathway Studies: Recombinant HTRA2 can be used in cell-free apoptosis reconstitution systems to evaluate its ability to cleave IAP substrates or to compete with endogenous IAP-binding proteins. When using cell lysates, ensure endogenous HTRA2 is adequately controlled.

Western Blot Positive Control: The recombinant protein serves as a molecular-weight and immunoreactivity positive control for anti-HTRA2 antibodies. Load 50–100 ng per lane alongside experimental cell lysates.

Protein Interaction Studies (Co-IP / Pull-down): The PDZ domain of HTRA2 mediates interactions with XIAP and other substrates. Recombinant protein can be used in pull-down assays with GST-tagged or His-tagged partner proteins under native conditions. Avoid denaturing conditions that disrupt the PDZ fold.

Important Considerations: HTRA2 is a biologically active protease; handle with care to avoid adventitious proteolysis of other proteins in the sample. Keep working solutions on ice during experiments. Avoid sodium azide in buffers if performing activity assays, as it can interfere with some fluorogenic readouts.

Troubleshooting Guide

Problem	Possible Cause	Recommended Action
No detectable protease activity	Protein denatured during shipping or reconstitution; inhibitor carryover in buffer	Confirm cold-chain integrity; re-reconstitute with fresh buffer; ensure no PMSF in assay buffer
Visible precipitate after reconstitution	Protein aggregation due to rapid dissolution or suboptimal buffer	Centrifuge at 10,000 × g, 4°C, 5 min; use supernatant; try adding 0.1% BSA to buffer
Weak or absent western blot band	Insufficient protein loaded; antibody incompatibility; transfer failure	Increase loading to 100–200 ng; verify antibody lot and conditions; check transfer efficiency with Ponceau S
Inconsistent results between experiments	Repeated freeze-thaw cycles; pipetting variability at low concentrations	Use single-use aliquots; prepare working dilutions fresh; pre-wet pipette tips
High background in activity assay	Substrate auto-hydrolysis; contaminant proteases in reagents	Include substrate-only blank; use highest-grade BSA; filter-sterilize all buffers

Frequently asked questions

What is the recommended reconstitution buffer for Recombinant Human HTRA2 Protein?

The recommended reconstitution buffer and volume are specified on the Certificate of Analysis (CoA) supplied with each lot of rp365977. Typically, sterile PBS (pH 7.4) supplemented with 0.1% BSA is suitable for most applications; however, always consult the lot-specific CoA for confirmed recommendations before reconstitution.

How should Recombinant Human HTRA2 Protein be stored to maintain activity?

Lyophilized protein should be stored at -20°C to -80°C in a frost-free freezer. Once reconstituted, divide the protein into single-use aliquots and snap-freeze on dry ice or liquid nitrogen before storing at -80°C. Avoid repeated freeze-thaw cycles, as each cycle can significantly reduce serine protease activity.

Is Recombinant Human HTRA2 Protein suitable for use as a western blot positive control?

Yes, Recombinant Human HTRA2 Protein (rp365977) is well-suited as a positive control for western blot experiments using anti-HTRA2 antibodies. Load 50–100 ng per lane to obtain a clear band at the expected molecular weight. Ensure the antibody has been validated against the specific tag or the mature form of HTRA2 present in the recombinant product.

Can Recombinant Human HTRA2 Protein be used in cell-based apoptosis assays?

Recombinant Human HTRA2 Protein is primarily designed for cell-free biochemical assays, including protease activity assays and protein interaction studies. For cell-based applications, delivery of recombinant protein into intact cells typically requires specialized transfection or microinjection methods. Researchers should optimize delivery conditions and validate intracellular activity independently.

What quality control tests are performed on each lot of rp365977?

Quality control information including purity assessment (typically by SDS-PAGE or HPLC), protein concentration, and biological activity data is reported on the lot-specific Certificate of Analysis. Please contact Aladdin Scientific customer service or refer to the CoA included with your shipment for detailed lot-specific QC data.

References

1. Verhagen AM, et al. Identification of DIABLO, a mammalian protein that promotes apoptosis by binding to and antagonizing IAP proteins. Cell. 2000;102(1):43-53.
2. Martins LM, et al. The serine protease Omi/HtrA2 regulates apoptosis by binding XIAP through a reaper-like motif. J Biol Chem. 2002;277(1):439-444.
3. Suzuki Y, et al. A serine protease, HtrA2, is released from the mitochondria and interacts with XIAP, inducing cell death. Mol Cell. 2001;8(3):613-621.
4. Aladdin Scientific. Recombinant Human HTRA2 Protein, SKU rp365977. Product datasheet reference absea#PP-1616.

Products are supplied for research and development use only. Not for use in humans, animals, diagnosis, or therapy.