HTRA2 Antibody

What is HTRA2 and why is it important in research?

HTRA2 (also known as OMI or PRSS25) is a mitochondrial serine protease with versatile biological functions ranging from apoptosis regulation to maintaining neuronal cell survival and mitochondrial homeostasis. The protein contains an N-terminal mitochondrial targeting sequence (MTS), a transmembrane domain (TM), a central protease domain, a C-terminal PDZ domain, and an unconventional IAP-binding motif . HTRA2 is particularly significant in research because:

Loss of HTRA2 protease function is associated with neurodegeneration, while overactivation of its proteolytic function is linked to cell death and inflammation . Studies have shown increased HTRA2 protein activity in brain tissues of Alzheimer's disease patients, suggesting a neuroprotective role through enhancement of autophagic processes . Furthermore, HTRA2 promotes the degradation of mutant proteins like A53T α-synuclein through autophagy and might be involved in amyloid plaque removal . The mnd2 (motor neuron degeneration) mice with inactivating mutations in the HTRA2 protease domain exhibit muscle wasting and neurodegeneration, while HtrA2 knockout mice display neuronal degeneration and a parkinsonian phenotype .

These diverse functions and disease associations make HTRA2 a compelling target for researchers studying neurodegenerative diseases, apoptosis, and mitochondrial function.

What types of HTRA2 antibodies are available and how should they be selected for specific applications?

Various HTRA2 antibodies are available targeting different regions of the protein and optimized for different experimental applications. Selection should be based on the specific research needs:

Antibody Types by Target Region:

• C-Terminal targeting antibodies (e.g., antibodies against AA 278-458, AA 334-458, AA 359-458)

• N-Terminal targeting antibodies (e.g., antibodies against AA 73-102)

• Mid-region targeting antibodies (e.g., antibodies against AA 231-330)

Antibody Types by Host and Format:

• Mouse monoclonal antibodies (clones include 6H7A8, 4F10, 18-01-83, 8G11-4B8-4F10-G5, 196C429, E-3)

• Rabbit polyclonal antibodies (various options available)

When selecting an antibody, researchers should consider:

• The specific epitope being targeted (which may affect detection of different HTRA2 isoforms)

• The experimental application (WB, IHC, IP, ELISA, IF)

• Species reactivity (human, mouse, rat)

• The format (conjugated vs. unconjugated)

For example, when studying the cleaved, active form of HTRA2 released from mitochondria, C-terminal antibodies may be more appropriate as this region remains intact after processing. For co-immunoprecipitation studies, antibodies validated for IP applications should be selected .

How can I validate the specificity of an HTRA2 antibody for my experimental system?

Validating antibody specificity is crucial for obtaining reliable results. For HTRA2 antibodies, consider these methodological approaches:

Positive and Negative Controls:

• Positive control: Use tissues or cell lines known to express HTRA2 (most human cells express HTRA2, with higher levels in metabolically active tissues)

• Negative control: Use HTRA2 knockout cells/tissues where available, or tissues known to have very low expression

Molecular Weight Verification:

• Full-length HTRA2 is approximately 46 kDa before mitochondrial import

• Upon mitochondrial import and processing, HTRA2 is cleaved to yield products of approximately 37 and 35 kDa

• Verify that your antibody detects bands at these expected molecular weights

Peptide Competition Assay:

• Pre-incubate the antibody with the immunizing peptide (if available)

• Loss of signal in Western blot or immunostaining confirms specificity

siRNA/shRNA Knockdown:

• Transfect cells with HTRA2-specific siRNA/shRNA

• Reduction in signal intensity proportional to knockdown efficiency confirms specificity

Multiple Antibody Verification:

• Use multiple antibodies targeting different epitopes of HTRA2

• Consistent results across different antibodies increase confidence in specificity

Each validation method provides complementary information, and combining multiple approaches provides the strongest evidence for antibody specificity.

How can HTRA2 antibodies be used to investigate HTRA2's role in neurodegenerative diseases?

HTRA2 has been implicated in several neurodegenerative diseases, particularly Parkinson's disease (PD) and Alzheimer's disease (AD). HTRA2 antibodies can be instrumental in investigating these connections through various methodological approaches:

Tissue Expression Analysis:

• Immunohistochemistry (IHC) using HTRA2 antibodies can reveal altered expression patterns in neurodegenerative disease tissues compared to healthy controls

• Quantitative Western blotting can measure HTRA2 expression levels in different brain regions affected by neurodegenerative diseases

Post-translational Modification Analysis:

• Phospho-specific antibodies can detect specific modifications of HTRA2 associated with disease states

• Co-immunoprecipitation followed by mass spectrometry can identify disease-specific modifications

Protein-Protein Interaction Studies:

• Co-immunoprecipitation using HTRA2 antibodies can identify interaction partners in disease contexts

• Research has shown that HTRA2 has complex protein interaction networks in neurological tissues, with many interactors involved in ER to Golgi anterograde transport (e.g., AP3D1), aggrephagy (e.g., PSMC1), and pyruvate metabolism/citric acid cycle (e.g., SHMT2)

• Changes in these interaction networks in disease states may reveal pathological mechanisms

Subcellular Localization Studies:

• Immunofluorescence with HTRA2 antibodies can detect abnormal translocation from mitochondria in disease models

• This is particularly relevant as HTRA2 release from mitochondria is associated with apoptotic signaling

In Vivo and Ex Vivo Models:

• HTRA2 antibodies can be used to characterize disease phenotypes in animal models of neurodegeneration

• For example, HtrA2 null mutants exhibit mild mitochondrial defects, loss of flight and climbing ability, and sensitivity to oxidative stress and mitochondrial toxins, sharing phenotypic characteristics with Drosophila models of PD

These approaches can provide insights into how HTRA2 dysfunction contributes to neurodegenerative processes and identify potential therapeutic targets.

What methodologies can be used to study HTRA2 protease activity and how can antibodies complement these approaches?

Studying HTRA2 protease activity is crucial for understanding its biological functions. Several methodologies can be used, with antibodies playing complementary roles:

Fluorogenic Substrate Assays:

• HTRA2 protease activity can be measured using fluorogenic peptide substrates such as the H2-optimal substrate

• This substrate contains a quencher and fluorophore unit, where cleavage disrupts the quencher/fluorophore complex, allowing fluorescence measurement

• The assay is typically performed with recombinant HTRA2 (100 nM) in a protease assay buffer (50 mM TRIS, 0.5 mM EDTA, 1 mM DTT, pH 8.0) containing 10 μM H2-optimal substrate

• Fluorescence is monitored over time to determine the enzymatic reaction rate

Protease Inhibitor Studies:

• Inhibitors like UCF-101 (typically used at 30 μM) specifically target the catalytic domain of HTRA2

• Synthetic peptides like ASGYTFTNYGLSWVR (CDR1 peptide) have been shown to inhibit HTRA2 activity

• Antibodies can be used to verify inhibitor specificity through co-immunoprecipitation experiments

Co-immunoprecipitation of HTRA2 Substrates:

• HTRA2 antibodies can be used to immunoprecipitate HTRA2 along with its bound substrates

• Mass spectrometry analysis of the immunoprecipitated complexes can identify novel substrates

• Changes in substrate profiles under different conditions (e.g., disease states, stress conditions) can reveal context-specific functions

In-gel Zymography:

• HTRA2 can be separated by non-denaturing PAGE containing a substrate (e.g., casein)

• After electrophoresis, the gel is incubated to allow proteolysis, then stained to visualize areas of substrate degradation

• Western blotting with HTRA2 antibodies on parallel gels can confirm the identity of the proteolytic bands

Cellular Assays:

• Immunocytochemistry with HTRA2 antibodies can detect HTRA2 translocation from mitochondria during apoptosis

• Live-cell imaging combined with immunofluorescence can track dynamic changes in HTRA2 localization and correlate with protease activity

These complementary approaches provide a comprehensive understanding of HTRA2 protease activity in different contexts.

How can I optimize co-immunoprecipitation protocols to study HTRA2 protein interactions using HTRA2 antibodies?

Co-immunoprecipitation (Co-IP) is a powerful technique to study HTRA2 protein interactions. Based on published methodologies, here are optimized protocols and considerations:

Antibody Selection:

• Choose antibodies specifically validated for immunoprecipitation (IP)

• Consider using antibodies targeting different epitopes for confirmation, as some interactions might be masked by antibody binding

• For tagged recombinant HTRA2, consider tag-specific antibodies (e.g., His-tag antibodies for 6xHis-tagged HTRA2)

Sample Preparation:

• For tissue samples: Homogenize tissue in a gentle lysis buffer (e.g., PBS with protease inhibitors) to preserve protein-protein interactions

• For cell cultures: Lyse cells in non-denaturing buffers (e.g., RIPA buffer with reduced detergent concentration)

• Maintain low temperatures (4°C) throughout to preserve interactions

• Include protease and phosphatase inhibitors to prevent degradation and modification of interaction partners

Pre-clearing:

• Pre-clear lysates with protein A/G beads to reduce non-specific binding

• This step is especially important when working with complex tissue samples like brain or retina

Immunoprecipitation:

• For direct IP: Incubate clarified lysate with HTRA2 antibody (typically 2-5 μg per mg of total protein) followed by protein A/G beads

• For recombinant His-tagged HTRA2: Spike recombinant protein (e.g., 2 μg) into homogenized tissue (e.g., 5 mg) and capture using Ni-NTA magnetic beads (e.g., 40 μL)

• Incubate overnight at 4°C with gentle rotation to maximize binding while minimizing degradation

Washing and Elution:

• Wash beads 2-4 times with PBS or appropriate buffer to remove non-specific binders

• For His-tagged HTRA2, elute bound proteins by pH shift or with imidazole

• For antibody-based IP, elute with low pH buffer or by boiling in SDS sample buffer

Controls:

• Negative control: Perform parallel IP with isotype-matched control antibody or with lysate not containing HTRA2

• Specificity control: Include conditions with HTRA2 inhibitors (e.g., UCF-101) or competitors (e.g., synthetic CDR peptide) to identify specific versus non-specific interactions

Analysis:

• Analyze eluates by mass spectrometry for unbiased interaction partner identification

• Confirm key interactions by Western blotting with specific antibodies

• Compare interaction profiles under different conditions (e.g., with/without inhibitors, disease versus healthy state)

Following this optimized protocol can reveal complex protein interaction networks of HTRA2 in various tissues and conditions.

What are the technical considerations when using HTRA2 antibodies for immunohistochemistry in neurodegenerative disease tissues?

Immunohistochemistry (IHC) with HTRA2 antibodies in neurodegenerative disease tissues requires special considerations to obtain reliable and informative results:

Tissue Fixation and Processing:

• HTRA2 is a mitochondrial protein that can relocalize during apoptosis, so fixation method is critical

• For formalin-fixed paraffin-embedded (FFPE) tissues, optimize fixation time to prevent overfixation which may mask epitopes

• For frozen sections, use gentle fixation (e.g., 4% paraformaldehyde for 10-15 minutes) to preserve antigenicity

• Consider using both FFPE and frozen section approaches as complementary methods

Antigen Retrieval:

• HTRA2 epitopes may be masked during fixation, requiring antigen retrieval

• Test both heat-induced epitope retrieval (HIER) methods (e.g., citrate buffer pH 6.0, EDTA buffer pH 9.0) and enzymatic methods (e.g., proteinase K)

• Optimize retrieval conditions for your specific antibody and tissue type

Antibody Selection and Validation:

• Select antibodies specifically validated for IHC applications

• Validate antibody specificity in your tissue of interest using positive and negative controls

• Consider using multiple antibodies targeting different epitopes to confirm staining patterns

Background Reduction:

• Block endogenous peroxidase activity (for HRP-based detection) or endogenous biotin (for biotin-based detection)

• Use tissue-matched blocking serum containing the same species as the secondary antibody

• Include detergents (e.g., 0.1-0.3% Triton X-100) in antibody diluents to reduce non-specific membrane binding

• Consider using specialized blocking reagents for tissues with high background (e.g., brain tissues)

Signal Amplification and Detection:

• For low abundance targets, use signal amplification methods (e.g., tyramide signal amplification)

• When studying co-localization, use fluorescent secondary antibodies with spectrally distinct fluorophores

• For quantitative analysis, use automated image analysis software with appropriate controls

Interpretation Challenges in Neurodegenerative Tissues:

• Neurodegenerative tissues often contain protein aggregates that can trap antibodies non-specifically

• Include controls using pre-immune serum or isotype control antibodies

• Compare staining patterns with other mitochondrial markers to confirm subcellular localization

• When studying diseased tissue, include age-matched controls and analyze multiple brain regions

Quantification Approaches:

• Use digital image analysis for objective quantification of HTRA2 expression levels

• Normalize HTRA2 staining to appropriate housekeeping proteins or mitochondrial markers

• Consider semi-quantitative scoring systems (e.g., H-score) for comparative studies across disease stages

These technical considerations will help ensure reliable and informative results when using HTRA2 antibodies for IHC in neurodegenerative disease tissues.

How should experiments be designed to investigate HTRA2 antibody cross-reactivity with other HtrA family members?

HTRA2 belongs to the HtrA family of serine proteases, which in humans includes HTRA1, HTRA2, HTRA3, and HTRA4. Designing experiments to assess potential cross-reactivity requires systematic approaches:

Sequence Analysis and Epitope Mapping:

• Perform sequence alignment of all HtrA family members to identify regions of high homology

• Map the antibody epitope location and assess homology with corresponding regions in other HtrA proteins

• Use epitope prediction algorithms to identify potential cross-reactive epitopes

Recombinant Protein Testing:

• Express and purify all HtrA family members as recombinant proteins

• Perform Western blot analysis with the HTRA2 antibody against all family members

• Quantify relative binding affinity to determine cross-reactivity levels

Knockout/Knockdown Validation:

• Use HTRA2 knockout or knockdown cell lines as negative controls

• Test whether the antibody produces signals in these systems (which would indicate cross-reactivity)

• Additionally, overexpress other HtrA family members in these systems to directly assess cross-reactivity

Immunoprecipitation-Mass Spectrometry:

• Perform immunoprecipitation with the HTRA2 antibody

• Analyze the precipitated proteins by mass spectrometry

• Identify any other HtrA family members in the precipitate

Immunohistochemistry with Specific Controls:

• Perform IHC on tissues with known expression patterns of different HtrA family members

• Compare with IHC using validated antibodies specific for other HtrA proteins

• Include tissues from HTRA2 knockout animals as controls

Peptide Competition Assays:

• Design peptides corresponding to the epitope regions of all HtrA family members

• Perform competition assays to determine whether peptides from other family members can block antibody binding

Reporting Standards:

• Document all cross-reactivity testing methodologies in publications

• Provide quantitative assessments of cross-reactivity where possible

• Clearly state the limitations of the antibody regarding potential cross-reactivity

This systematic approach will provide comprehensive information about potential cross-reactivity, allowing researchers to make informed decisions about antibody applications and data interpretation.

What are the optimal protocols for using HTRA2 antibodies to study HTRA2's role in mitochondrial function and dysfunction?

HTRA2's localization in mitochondria and its role in mitochondrial homeostasis make it an important target for studying mitochondrial function. Here are optimized protocols using HTRA2 antibodies:

Subcellular Fractionation and Western Blotting:

• Isolate mitochondrial, cytosolic, and nuclear fractions using differential centrifugation

• Verify fraction purity using markers like VDAC (mitochondria), GAPDH (cytosol), and Lamin B (nucleus)

• Perform Western blotting with HTRA2 antibodies to track HTRA2 localization under normal and stress conditions

• Quantify the ratio of mitochondrial to cytosolic HTRA2 as an indicator of mitochondrial release

Mitochondrial Morphology Assessment:

• Perform immunofluorescence co-staining with HTRA2 antibodies and mitochondrial markers (e.g., MitoTracker, TOM20)

• Use confocal microscopy to analyze mitochondrial morphology parameters (length, fragmentation, network connectivity)

• Compare these parameters in cells with normal versus altered HTRA2 expression/activity

• HtrA2 knockout mice exhibit abnormal mitochondria, suggesting HTRA2's importance in maintaining mitochondrial integrity

Mitochondrial Membrane Potential:

• Use potential-sensitive dyes (e.g., JC-1, TMRM) in combination with immunofluorescence for HTRA2

• Correlate HTRA2 expression/localization with membrane potential changes

• Perform time-lapse imaging to track dynamic changes during cellular stress

Mitochondrial Respiration Analysis:

• Measure oxygen consumption rate (OCR) using platforms like Seahorse XF Analyzer

• Compare OCR in cells with normal, depleted, or inhibited HTRA2

• Use HTRA2 antibodies to verify HTRA2 status in parallel samples

• HtrA2 mutants exhibit mitochondrial defects that could affect respiratory function

Mitochondrial Stress Response:

• Induce mitochondrial stress with toxins like rotenone or CCCP

• Track HTRA2 expression, processing, and localization using specific antibodies

• Correlate with markers of mitochondrial unfolded protein response (UPRmt)

• HtrA2 null mutants show sensitivity to oxidative stress and mitochondrial toxins

Mitophagy Assessment:

• Co-stain for HTRA2 and mitophagy markers (e.g., PINK1, Parkin, LC3)

• Track co-localization during mitophagy induction

• Use live-cell imaging to monitor dynamics of HTRA2-positive mitochondria during mitophagy

Electron Microscopy with Immunogold Labeling:

• Perform immunogold labeling with HTRA2 antibodies for transmission electron microscopy

• Analyze ultrastructural localization of HTRA2 within mitochondria

• Compare mitochondrial ultrastructure in normal versus disease models

These protocols provide complementary approaches to study HTRA2's role in mitochondrial function and dysfunction, particularly relevant for neurodegenerative disease research.

How can researchers troubleshoot common problems when using HTRA2 antibodies in Western blotting applications?

Western blotting with HTRA2 antibodies can present several challenges. Here are methodological solutions to common problems:

Problem: No signal or weak signal

Potential causes and solutions:

• Insufficient HTRA2 expression: Confirm HTRA2 expression in your sample using RT-PCR or enriched mitochondrial fractions

• Antibody dilution too high: Perform titration experiments to determine optimal antibody concentration

• Inefficient protein transfer: Verify transfer efficiency with Ponceau S staining; consider optimizing transfer conditions for mitochondrial proteins

• Epitope masking: Try different extraction buffers; HTRA2 is a mitochondrial protein that may require specialized extraction methods

• Epitope destruction during processing: Avoid excessive sample heating; include protease inhibitors in all buffers

Problem: Multiple unexpected bands

Potential causes and solutions:

• Cross-reactivity: Verify specificity using HTRA2 knockdown/knockout controls

• HTRA2 processing products: HTRA2 exists in different forms (full-length ~46 kDa; processed forms ~37 and 35 kDa) ; confirm band identity using recombinant standards

• Protein degradation: Include fresh protease inhibitors in all buffers; keep samples cold throughout preparation

• Non-specific secondary antibody binding: Include proper blocking agents; test alternative secondary antibodies

Problem: High background

Potential causes and solutions:

• Insufficient blocking: Increase blocking time/concentration; test alternative blocking agents (e.g., BSA vs. milk)

• Antibody concentration too high: Dilute primary and/or secondary antibodies

• Membrane overexposure: Reduce exposure time during imaging

• Detergent concentration: Optimize wash buffer detergent concentration (typically 0.05-0.1% Tween-20)

Problem: Inconsistent results between experiments

Potential causes and solutions:

• HTRA2 expression variability: Standardize culture/treatment conditions; use appropriate housekeeping controls

• Antibody batch variation: Use the same lot when possible; include positive controls with each experiment

• Sample preparation inconsistency: Standardize protein extraction and quantification protocols

• Loading control issues: Verify equal loading with total protein stains (e.g., Ponceau S, REVERT)

Problem: Discrepancy between results with different HTRA2 antibodies

Potential causes and solutions:

• Different epitopes: Different antibodies may recognize different forms of HTRA2; map the epitopes to understand what each antibody should detect

• Isoform specificity: Verify which isoforms each antibody can detect

• Post-translational modifications: Some antibodies may be sensitive to modifications that mask epitopes

• Specificity differences: Validate each antibody using knockdown/knockout controls

Recommended optimization strategy:

• Start with positive control samples known to express HTRA2 (e.g., HeLa cells)

• Use mitochondrial enrichment to increase signal-to-noise ratio

• Test multiple antibody dilutions (typically 1:500 to 1:5000)

• Include appropriate molecular weight markers to identify specific HTRA2 forms

• Document all conditions systematically to identify optimal parameters

Following these troubleshooting strategies will help researchers obtain reliable and reproducible Western blot results with HTRA2 antibodies.

How should researchers interpret contradictory results between different HTRA2 antibodies in studies of neurodegenerative diseases?

Contradictory results between different HTRA2 antibodies in neurodegenerative disease studies require systematic investigation and careful interpretation:

Sources of Contradictions:

• Epitope-specific differences:

  • Different antibodies target different regions of HTRA2

  • Some epitopes may be masked or modified in disease states

  • Solution: Map the exact epitopes of each antibody to understand what each is detecting

• Disease-specific modifications:

  • Neurodegenerative diseases may induce post-translational modifications or conformational changes

  • These changes may affect epitope accessibility differentially

  • Solution: Use techniques like mass spectrometry to identify disease-specific modifications

• Processing differences:

  • HTRA2 undergoes processing from a ~46 kDa precursor to ~37 and 35 kDa mature forms

  • Disease states may alter processing efficiency

  • Solution: Use antibodies specific to different regions to track processing status

• Subcellular localization changes:

  • HTRA2 can relocalize from mitochondria during stress/disease

  • Solution: Use subcellular fractionation and localization studies with multiple antibodies

Methodological Approach to Resolve Contradictions:

• Comprehensive antibody validation:

  • Validate each antibody using knockout/knockdown controls

  • Confirm specificity with peptide competition assays

  • Test reactivity against recombinant HTRA2 protein

• Multi-technique confirmation:

  • Use complementary techniques (Western blot, IHC, IP-MS)

  • Compare results across techniques for each antibody

  • Consider that some antibodies may work better in certain applications

• Control experiments:

  • Include tissue from models with altered HTRA2 (e.g., HtrA2 null mutants)

  • Use tissues with known HTRA2 alterations as positive controls

• Careful sample handling:

  • Standardize tissue collection, processing, and storage

  • Process disease and control samples identically

  • Document post-mortem intervals for human samples

Interpretation Framework:

• When antibodies targeting different domains show different results:

  • Consider domain-specific modifications or interactions

  • Investigate whether disease-associated mutations affect specific domains

  • Examine whether proteolytic processing differs in disease tissues

• When different techniques yield contradictory results:

  • Consider technique-specific limitations (e.g., epitope masking in FFPE tissues)

  • Evaluate whether sample preparation affects HTRA2 structure differently

  • Determine which technique provides the most relevant biological context

• When results differ between disease models and human samples:

  • Consider species-specific differences in HTRA2 sequence and processing

  • Evaluate whether model systems accurately recapitulate human disease aspects

  • Assess whether disease duration/severity affects results

• Data reporting best practices:

  • Report results from multiple antibodies separately rather than combining

  • Clearly document which antibody was used for each experiment

  • Discuss limitations and potential reasons for contradictions

  • Consider publishing null or contradictory results to advance the field

By following this systematic approach, researchers can transform contradictory results into valuable insights about disease-specific changes in HTRA2 biology.