HAO1 Monoclonal Antibody

What is HAO1 and what is its primary function in cellular metabolism?

HAO1 (Hydroxyacid oxidase 1) is a member of the FMN-dependent alpha-hydroxy acid dehydrogenase family with a molecular weight of approximately 40924 Da . It functions primarily as a peroxisomal enzyme that catalyzes the oxidation of 2-hydroxyacids, with particular activity toward glycolate (a two-carbon substrate) and 2-hydroxy fatty acids . The enzyme plays a critical role in glyoxylate metabolism and contributes to cellular redox homeostasis. HAO1 is one of three related genes that have 2-hydroxyacid oxidase activity yet differ in encoded protein amino acid sequence, tissue expression, and substrate preference .

What is the tissue distribution pattern of HAO1 expression?

HAO1 is expressed predominantly in the liver and pancreas, with particularly high levels detected in hepatocytes . The protein localizes to peroxisomes, where it participates in metabolic pathways involving glycolate oxidation . Interestingly, the transcript detected at high levels in the pancreas may represent an alternatively spliced form or result from the use of multiple near-consensus upstream polyadenylation sites . When designing experiments to study HAO1, researchers should consider this tissue-specific expression pattern and select appropriate positive control tissues for validation studies.

What are the known alternative names and nomenclature for HAO1?

When searching literature and databases, researchers should be aware of the various nomenclature used for HAO1, including:

• HAOX1 (Hydroxyacid oxidase 1)

• GOX (Glycolate oxidase)

• GOX1 (Glycolate oxidase 1)

• Hao-1

This awareness is particularly important when conducting comprehensive literature reviews or database searches to ensure all relevant research is captured.

How do monoclonal and polyclonal HAO1 antibodies differ in research applications?

Monoclonal HAO1 antibodies, typically mouse-derived (as seen in products from Boster Bio and Abbkine), offer high specificity to single epitopes, providing consistent results across experimental replicates and reducing background noise in techniques like Western blotting . These antibodies are particularly valuable for targeted detection of specific HAO1 domains or isoforms.

Conversely, polyclonal HAO1 antibodies (such as the rabbit polyclonal offered by Assay Genie) recognize multiple epitopes on the HAO1 protein, potentially enhancing signal strength in applications like immunohistochemistry where antigen retrieval might damage some epitopes . This multi-epitope recognition can be advantageous for detecting proteins expressed at low levels.

Selection between monoclonal and polyclonal antibodies should be guided by experimental requirements, with monoclonals preferred for studies requiring high specificity to particular domains and polyclonals for applications benefiting from enhanced signal amplification.

What species reactivity is available for commercial HAO1 monoclonal antibodies?

Commercial HAO1 monoclonal antibodies demonstrate varied species reactivity profiles:

Researchers should carefully select antibodies with validated reactivity to their species of interest . When working with non-validated species, preliminary titration experiments and positive controls are strongly recommended to confirm cross-reactivity.

What are the optimal storage conditions for preserving HAO1 antibody activity?

HAO1 monoclonal antibodies require specific storage conditions to maintain their functional activity:

• Long-term storage: -20°C for up to one year

• Short-term storage (frequent use): 4°C for up to one month

• Storage buffer composition: PBS (pH 7.4) containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide as preservative

To maximize antibody shelf-life and performance:

• Avoid repeated freeze-thaw cycles by preparing small working aliquots

• Centrifuge the original vial after thawing and prior to removing the cap to recover maximum product

• When diluting for experiments, use sterile techniques and appropriate diluents that maintain protein stability

What are the validated applications for HAO1 monoclonal antibodies?

HAO1 monoclonal antibodies have been validated for multiple experimental applications:

For optimal results in each application, researchers should:

• Include positive control tissues (liver being optimal)

• Perform preliminary titration experiments to determine optimal antibody concentration

• Include appropriate negative controls (secondary antibody only, isotype controls)

What protocol modifications are recommended for optimal HAO1 detection in Western blot applications?

For optimal HAO1 detection in Western blot applications:

• Sample preparation:

  • Use RIPA buffer supplemented with protease inhibitors

  • For enriched detection, consider peroxisomal isolation protocols

  • Load 20-40 μg of total protein from liver lysates (primary expressing tissue)

• Gel electrophoresis:

  • 10-12% SDS-PAGE gels provide optimal resolution for the ~41 kDa HAO1 protein

  • Include positive controls (liver tissue) and molecular weight markers

• Antibody incubation:

  • Primary antibody: Use 1:1000-2000 dilution in 5% BSA/TBST

  • Incubate overnight at 4°C for optimal sensitivity

  • Secondary antibody: HRP-conjugated anti-mouse IgG at 1:5000

• Detection optimization:

  • Enhanced chemiluminescence (ECL) provides sufficient sensitivity

  • For low-expressing samples, consider ECL-Plus or fluorescent secondary antibodies

How can HAO1 antibodies be utilized in immunohistochemistry for different tissue types?

Optimized IHC-P protocols for HAO1 detection across various tissues:

• Tissue preparation:

  • 4% paraformaldehyde fixation followed by paraffin embedding

  • 4-6 μm section thickness is optimal

  • Heat-mediated antigen retrieval in citrate buffer (pH 6.0) is recommended

• Tissue-specific considerations:

  • Liver tissue: Primary antibody at 1:200 dilution shows strong peroxisomal staining pattern

  • Kidney tissue: 1:200 dilution with extended antigen retrieval (20 minutes)

  • Colon tissue: 1:200 dilution with detection enhancement using polymer-HRP systems

  • Uterine cancer tissue: 1:200 dilution with dual antigen retrieval (heat + enzymatic)

• Detection systems:

  • DAB chromogen provides strong signal for liver samples

  • For tissues with lower expression, consider tyramide signal amplification

  • Counterstain with hematoxylin for 30-60 seconds for optimal nuclear contrast

How can researchers troubleshoot weak or nonspecific signals when using HAO1 antibodies?

When encountering weak or nonspecific signals, implement these methodological approaches:

• Weak or no signal:

  • Increase antibody concentration incrementally (start with 2-fold increase)

  • Extend primary antibody incubation time to overnight at 4°C

  • Verify tissue expression patterns (liver and pancreas show highest expression)

  • Implement signal enhancement techniques (TSA systems, more sensitive ECL substrates)

  • Confirm sample integrity with housekeeping protein detection

• Nonspecific or high background signals:

  • Increase blocking stringency (5% BSA with 0.1% Tween-20)

  • Reduce primary antibody concentration

  • Include additional washing steps (5 × 5 minutes)

  • Use more dilute secondary antibody

  • For IHC/IF: Include tissue-specific blocker (normal serum from secondary antibody host species)

  • Confirm specificity through peptide competition or knockout/knockdown controls

What cross-reactivity considerations should researchers address when using HAO1 antibodies?

HAO1 belongs to a family of hydroxyacid oxidases that share sequence homology. To address potential cross-reactivity:

• Sequence homology assessment:

  • HAO1 shares structural features with HAO2 and HAO3 family members

  • Verify the immunogen sequence used for antibody generation against other family members

  • The immunogen containing amino acids 1-370 of human HAO1/GOX (NP_060015.1) should be assessed for uniqueness

• Experimental validation approaches:

  • Include tissues known to express related family members as specificity controls

  • Consider parallel detection with antibodies targeting different epitopes

  • In crucial experiments, validate findings with genetic approaches (siRNA, CRISPR)

  • Use recombinant HAO1, HAO2, and HAO3 proteins in dot blot or Western blot analysis to determine cross-reactivity profiles

How should researchers optimize fixation and permeabilization protocols for HAO1 detection in immunofluorescence?

HAO1's peroxisomal localization requires optimized protocols for immunofluorescence:

• Fixation optimization:

  • 4% paraformaldehyde (10-15 minutes at room temperature) preserves epitope accessibility

  • Avoid methanol fixation which can disrupt peroxisomal integrity

  • For double-labeling with peroxisomal markers, confirm compatibility of fixation methods

• Permeabilization strategies:

  • Triton X-100 (0.1-0.3%) for 5-10 minutes enables antibody access to peroxisomal proteins

  • Digitonin (50 μg/ml) provides selective permeabilization that preserves peroxisomal structure

  • Saponin (0.1%) offers reversible permeabilization that maintains organelle integrity

• Antigen retrieval considerations:

  • Heat-mediated antigen retrieval in citrate buffer (pH 6.0) enhances detection sensitivity

  • For co-localization studies with other peroxisomal proteins, verify compatible retrieval methods

• Mounting and visualization:

  • Use anti-fade mounting media without DAPI if performing spectral imaging

  • Super-resolution microscopy techniques (STED, SIM) can resolve individual peroxisomes

How can HAO1 antibodies be employed in studying oxidative stress mechanisms?

HAO1's role in glycolate metabolism produces hydrogen peroxide, connecting it to oxidative stress pathways. Advanced research applications include:

• Co-immunoprecipitation studies:

  • Use HAO1 monoclonal antibodies to isolate protein complexes

  • Identify interacting partners in oxidative stress response pathways

  • Protocol recommendation: Crosslink antibody to magnetic beads for cleaner precipitations

• Post-translational modification analysis:

  • Detect oxidative stress-induced modifications of HAO1

  • Combined approach using phospho-specific and HAO1 antibodies

  • Mass spectrometry validation of modification sites

• Peroxisomal dynamics during oxidative stress:

  • Co-labeling of HAO1 with peroxisomal membrane markers during stress conditions

  • Time-course analysis using live-cell imaging with tagged HAO1 constructs

  • Correlative light and electron microscopy to connect HAO1 distribution with ultrastructural changes

What methodological approaches can resolve contradictory findings in HAO1 research?

When facing contradictory findings about HAO1 function or expression:

• Antibody validation hierarchy:

  • Employ multiple antibodies targeting different epitopes

  • Validate with genetic models (knockout tissues, CRISPR-edited cells)

  • Complement with mRNA quantification and in situ hybridization

• Context-dependent expression analysis:

  • Developmental stage-specific expression patterns

  • Metabolic state-dependent regulation (fed vs. fasted)

  • Species-specific differences in expression and function

• Functional validation approaches:

  • Enzymatic activity assays paired with protein expression analysis

  • Metabolomic profiling to assess pathway impacts

  • Isotope tracing experiments to confirm metabolic flux

How can HAO1 antibodies contribute to understanding disease mechanisms beyond ectopic ossification?

Recent research indicates HAO1 is not a pathogenic factor for ectopic ossifications , but its involvement in other disease mechanisms warrants investigation:

• Metabolic disorder connections:

  • Quantitative analysis of HAO1 expression in models of diabetes and obesity

  • Subcellular distribution changes in metabolically stressed tissues

  • Correlation of HAO1 activity with glyoxylate pathway intermediates

• Cancer research applications:

  • Tissue microarray analysis across cancer types and grades

  • Assessment of HAO1 as biomarker through multiplexed IHC/IF

  • Correlation with metabolic reprogramming markers

• Neurodegenerative disease investigation:

  • HAO1 expression in models of oxidative stress-associated neurodegeneration

  • Co-localization with markers of peroxisomal dysfunction in patient samples

  • Temporal relationship between HAO1 dysregulation and disease progression

How can proximity labeling techniques enhance HAO1 interaction studies?

Proximity labeling offers advanced capabilities for studying HAO1 in its native environment:

• BioID and TurboID approaches:

  • Generate HAO1-BioID fusion constructs for expression in relevant cell types

  • Identify proximal proteins through streptavidin pulldown followed by mass spectrometry

  • Compare interactomes under normal vs. oxidative stress conditions

• APEX2-based proximity labeling:

  • HAO1-APEX2 constructs provide higher spatial and temporal resolution

  • Electron microscopy compatibility allows ultrastructural visualization

  • Recommended protocol modifications: Short labeling times (1-2 minutes) to capture dynamic interactions

• Data analysis considerations:

  • Filter against previous peroxisomal proximity labeling datasets

  • Validate top candidates through reciprocal labeling experiments

  • Functional clustering to identify novel pathway connections

What are the methodological considerations for multiplexed detection of HAO1 with other peroxisomal markers?

Advanced multiplexed detection requires careful methodological planning:

• Spectral imaging approaches:

  • Sequential detection using primary antibodies from different host species

  • Tyramide signal amplification with spectrally distinct fluorophores

  • Spectral unmixing algorithms to separate overlapping signals

• Cyclic immunofluorescence methods:

  • Antibody stripping and reprobing protocols optimized for peroxisomal antigens

  • Signal normalization between cycles using fiducial markers

  • Computational alignment of sequential imaging data

• Mass cytometry (CyTOF) for tissue analysis:

  • Metal-conjugated HAO1 antibodies combined with other peroxisomal markers

  • Single-cell resolution of peroxisomal protein abundance

  • Dimensionality reduction techniques to identify cellular subpopulations