HTRA1 Antibody, HRP conjugated

What is HTRA1 and why is it significant in research?

HTRA1 is a multidomain serine protease that belongs to the high-temperature requirement A (HtrA) family. It contains an N-terminal insulin-like growth factor binding protein (IGFBP) domain, a Kazal-type trypsin inhibitor motif, a trypsin-like protease domain, and a C-terminal PDZ domain . HTRA1 is extensively studied due to its involvement in:

• Age-related macular degeneration (AMD) pathogenesis and progression

• Regulation of TGF-beta signaling pathways

• Extracellular matrix protein degradation

• Tumor suppression activities

• Neurodegenerative diseases including Alzheimer's disease

• Arthritis (rheumatoid and osteoarthritis)

• Cerebral autosomal recessive arteriopathy (CARASIL)

HTRA1 functions as a protease that cleaves various substrates including extracellular matrix proteins like fibronectin, proteoglycans (aggrecan, decorin, fibromodulin), and insulin-like growth factor binding proteins . Through these activities, HTRA1 plays critical roles in tissue remodeling, growth factor availability, and cellular signaling.

What is the molecular structure and weight of HTRA1 protein?

When working with HTRA1 antibodies, understanding the protein's molecular characteristics is essential:

In Western blot applications, HTRA1 typically appears as a major band at approximately 51 kDa, with potential additional bands representing autolytic fragments or oligomeric forms .

What are the applications of HTRA1 antibody, HRP conjugated?

HRP-conjugated HTRA1 antibodies offer several advantages for research applications:

Optimal working dilutions for HRP-conjugated HTRA1 antibodies typically range from 1:500-1:1000 for Western blot and 1:50-1:500 for immunohistochemistry applications, though researchers should perform optimization for their specific experimental conditions .

How do I select appropriate positive and negative controls for HTRA1 antibody experiments?

Proper controls are essential for validating experimental results with HTRA1 antibodies:

Positive Controls:

• Human placenta tissue (high endogenous HTRA1 expression)

• JAR human choriocarcinoma cell line

• HeLa cells

• HepG2 cells (for immunofluorescence)

Negative Controls:

• HTRA1 knockout HAP1 cell lysates (validated control for specificity)

• Cell lines treated with HTRA1-specific siRNA (>90% reduction has been documented)

• Primary antibody omission control

• Isotype-matched irrelevant antibody control

For genetic validation, both HTRA1 knockout and knockdown approaches have been successfully employed, with knockout HAP1 cells showing complete loss of the expected 51 kDa band in Western blot applications .

How do I optimize Western blot protocols when using HTRA1 antibody, HRP conjugated?

Optimizing Western blot protocols for HTRA1 detection requires consideration of several technical factors:

Sample Preparation:

• Use PVDF membranes for optimal protein binding

• Test both reducing and non-reducing conditions (some epitopes may be masked under certain conditions)

• Include protease inhibitors in lysis buffers to prevent HTRA1 autolysis

• Load 20-30 μg of total protein for moderate HTRA1 expression

Protocol Optimization:

• Block with 5% non-fat milk in TBST (optimal for reducing background)

• Antibody dilution: Start with 1:500-1:1000 and adjust as needed

• Use Immunoblot Buffer Group 1 for optimal results with certain antibodies

• For challenging samples, consider overnight incubation at 4°C

Detection Strategies:

• Test different exposure times to capture optimal signal without saturation

• For low abundance detection, use enhanced chemiluminescence substrates

• Consider testing antibodies that target different epitopes of HTRA1 (e.g., N-terminal vs. C-terminal)

A systematic approach to optimization will yield consistent and specific detection of HTRA1 protein in your experimental system.

How can I validate the specificity of my HTRA1 antibody, HRP conjugated?

Antibody validation is critical for ensuring reliable research outcomes. For HTRA1 antibodies, multiple validation approaches should be employed:

Genetic Validation:

• Compare signal between wild-type and HTRA1 knockout cell lines (e.g., HAP1 knockout cell line)

• Use HTRA1-specific siRNA to knockdown expression (validated approaches have achieved >90% reduction)

• Overexpression studies with recombinant HTRA1

Analytical Validation:

• Peptide competition assays using the immunizing peptide/protein

• Test reactivity against specific domains/fragments of HTRA1

• Mass spectrometry confirmation of immunoprecipitated proteins

Functional Validation:

• Activity-based probe (ABP) competition assays to measure inhibition of HTRA1 activity

• Substrate cleavage assays to confirm functionality

Abcam's HTRA1 antibody (ab274322) demonstrates robust validation through knockout cell testing, showing complete loss of the 51 kDa band in HTRA1 knockout HAP1 cells .

What methodological approaches can I use to troubleshoot weak or no signal with HTRA1 antibody, HRP conjugated?

When encountering weak or absent signals with HTRA1 antibodies, implement this systematic troubleshooting workflow:

• Verify Antibody Activity:

  • Test with known positive control (human placenta tissue)

  • Check antibody storage conditions (-20°C, with 50% glycerol)

  • Verify HRP enzyme activity with direct substrate test

• Sample Quality Assessment:

  • Ensure proper protein extraction (test different lysis buffers)

  • Check for protein degradation (add fresh protease inhibitors)

  • Test sample preparation methods (non-reducing vs. reducing conditions)

• Protocol Optimization:

  • Increase antibody concentration (try 2-5× recommended dilution)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Use more sensitive detection reagents

  • Increase protein loading (50-75 μg total protein)

• Technical Considerations:

  • For Western blot: Verify transfer efficiency with reversible stain

  • For IHC/IF: Test different antigen retrieval methods (TE buffer pH 9.0 vs. citrate buffer pH 6.0)

  • For ELISA: Check plate binding capacity and blocking efficiency

A methodical approach to troubleshooting will help identify the source of the problem and guide appropriate solutions.

How do I interpret multiple bands or unexpected patterns in HTRA1 Western blots?

HTRA1 can exhibit complex banding patterns that require careful interpretation:

Expected Patterns:

• Primary band at approximately 51 kDa (full-length HTRA1)

• Lower molecular weight bands (35-50 kDa) may represent autolytic cleavage products

• Higher molecular weight bands (100-150 kDa) may indicate dimers or trimers

Interpretation Guidelines:

• Autolysis: HTRA1 undergoes self-cleavage, generating multiple fragments. This is particularly evident in samples with high HTRA1 expression or extended storage .

• Oligomerization: HTRA1 can form dimers and trimers, especially under non-reducing conditions, appearing as bands at 2-3× the monomeric molecular weight.

• Post-translational Modifications: Glycosylation or other modifications may cause shifts in apparent molecular weight.

• Sample Preparation Impact: Fresh vs. stored samples and reducing vs. non-reducing conditions significantly affect banding patterns.

For complex patterns, consider additional validation approaches such as mass spectrometry or antibodies targeting different epitopes to confirm band identity.

How can I use HTRA1 antibody, HRP conjugated, to study its role in age-related macular degeneration?

Studying HTRA1's role in AMD requires sophisticated experimental approaches:

Tissue Analysis:

• Perform comparative immunohistochemistry on normal vs. AMD-affected retinal tissues

• Quantify HTRA1 expression in different retinal layers

• Co-localize HTRA1 with drusen deposits and disease markers

Substrate Identification:

• Analyze HTRA1-specific substrates in ocular tissues using N-terminomics

• Focus on validated substrates such as DKK3, CLU, and RBP3

• Develop ELISAs for substrate cleavage products as biomarkers

Functional Assessment:

• Combine with activity-based probes (ABPs) to study active HTRA1 in vitreous samples

• Use ABP competition assays to measure inhibition by therapeutic antibodies

• Track pharmacodynamic biomarkers like DKK3 cleavage products

Therapeutic Development:

• Screen for HTRA1 inhibitory antibodies using activity assays

• Test efficacy of antibodies like Fab15H6.v4.D221 on HTRA1 activity

• Measure effects on substrate processing and disease progression

This multi-faceted approach provides comprehensive insights into HTRA1's contribution to AMD pathogenesis and potential therapeutic interventions.

How can I design experiments to evaluate HTRA1 inhibition using activity-based probes and HRP-conjugated antibodies?

Researchers can employ sophisticated methods to evaluate HTRA1 inhibition combining activity-based probes (ABPs) with antibody detection:

ABP Competition Assay Design:

• Pre-incubate HTRA1 with test inhibitors/antibodies at various concentrations

• Add HTRA1-specific ABP (e.g., with TAMRA fluorescent tag)

• Analyze by SDS-PAGE with fluorescence detection

• Calculate inhibition potency (IC50) relative to controls

Experimental Setup:

Readout and Analysis:

• Quantify fluorescent band intensity at 50 kDa (intact HTRA1)

• Plot inhibition curves (% activity vs. inhibitor concentration)

• Calculate IC50 values (35.9-51.0 nM reported for anti-HTRA1 Fab15H6.v4.D221)

This system has been successfully applied to measure HTRA1 inhibition in both buffer systems and complex biological matrices such as vitreous humor .

What methods can I use to identify and validate HTRA1 substrates using HRP-conjugated antibodies?

Comprehensive identification and validation of HTRA1 substrates requires integrating multiple techniques:

N-terminomics/TAILS Approach:

• Incubate biological samples with active HTRA1 vs. inactive HTRA1 or with/without inhibitory antibodies

• Identify neo-N-terminal peptides generated by HTRA1 cleavage using mass spectrometry

• Compare peptide profiles to identify HTRA1-specific cleavage sites

• Validate findings with synthetic peptide substrates

Western Blot Validation:

• Incubate purified candidate substrates with HTRA1

• Analyze cleavage patterns by Western blot using substrate-specific antibodies

• Confirm HTRA1-specific cleavage by including inhibitory antibodies

• Quantify cleavage efficiency using densitometry

In Vivo Substrate Validation:

• Administer HTRA1 inhibitory antibodies to animal models (e.g., intravitreal injection)

• Collect tissue samples (e.g., vitreous humor)

• Analyze substrate cleavage using Western blot or ELISA

• Compare substrate profiles in treated vs. control animals

Research has identified several validated HTRA1 substrates including:

• Dickkopf-related protein 3 (DKK3)

• Clusterin (CLU)

• Retinol-binding protein 3 (RBP3)

• Fibronectin

• Various proteoglycans

These substrates can serve as valuable biomarkers for HTRA1 activity in both preclinical and clinical studies.

How can I design experiments to study HTRA1's regulation of TGF-beta signaling using HRP-conjugated antibodies?

Investigating HTRA1's impact on TGF-beta signaling requires careful experimental design:

Receptor-Level Analysis:

• Treat cells with recombinant HTRA1 with/without inhibitory antibodies

• Immunoprecipitate TGF-beta receptors using specific antibodies

• Analyze receptor integrity/processing by Western blot with HRP-conjugated antibodies

• Quantify receptor degradation as a function of HTRA1 activity

Signaling Pathway Monitoring:

• Establish reporter cell lines (SMAD-responsive elements)

• Modulate HTRA1 levels (overexpression, knockdown, antibody inhibition)

• Stimulate with TGF-beta ligands

• Measure pathway activation (phospho-SMAD levels, reporter activity)

Multiplex Analysis Design:

This integrated approach provides mechanistic insights into how HTRA1 regulates TGF-beta signaling at multiple levels, from receptor processing to downstream transcriptional responses.

What experimental approaches can monitor HTRA1 activity in vivo using HRP-conjugated antibodies?

Monitoring HTRA1 activity in vivo requires sophisticated methods that balance sensitivity with specificity:

Intravitreal Administration Study Design:

• Administer anti-HTRA1 antibodies at various doses (0.001-6 mg per eye)

• Collect vitreous humor at different time points (1-14 days)

• Analyze HTRA1 activity ex vivo using activity-based probes

• Track substrate cleavage products as biomarkers

Activity Assessment Methods:

• Activity-Based Probe Analysis: Label collected samples with HTRA1-specific ABPs to quantify active enzyme

• Substrate Cleavage Monitoring: Analyze DKK3 processing as a pharmacodynamic biomarker

• Western Blot Analysis: Detect HTRA1 and substrate processing using HRP-conjugated antibodies

Species-Specific Considerations:

• Rabbit models show sustained HTRA1 inhibition at 0.02-2 mg doses for at least 14 days

• Cynomolgus monkeys exhibit complete HTRA1 inhibition at 0.02-6 mg doses throughout study duration

• Lower doses (0.001-0.002 mg) show transient inhibition with recovery by day 14

These approaches have successfully demonstrated dose-dependent and time-dependent inhibition of HTRA1 activity in vivo, providing crucial pharmacodynamic data for therapeutic development.