FAHD2A Antibody

What is FAHD2A and what are its primary functions?

FAHD2A (also known as CGI-105) is a member of the fumarylacetoacetate hydrolase family that may possess hydrolase activity . It is also identified as an oxaloacetate tautomerase (EC 5.3.2.2) with mitochondrial localization . The protein is encoded by the FAHD2A gene (Gene ID: 51011) located on chromosome 2q11.2 . The protein has a predicted molecular weight of approximately 34.6 kDa, though it is sometimes observed at around 31 kDa in Western blot analysis .

FAHD2A is involved in metabolic processes and belongs to the FAH family of proteins that typically participate in amino acid catabolism pathways . While its precise biological function is still being investigated, its hydrolase and tautomerase activities suggest roles in cellular metabolism.

Several types of FAHD2A antibodies are available for research purposes:

• Monoclonal antibodies: Produced from single B-cell clones, offering high specificity and consistency. Examples include:

  • Mouse monoclonal antibody clone OTI7B7

  • Mouse monoclonal antibody clone 7C9

  • Mouse monoclonal antibody clone 24GB13030

• Polyclonal antibodies: Produced from multiple B-cell lineages, recognizing multiple epitopes:

  • Rabbit polyclonal antibodies targeting various regions of FAHD2A

  • Mouse polyclonal antibodies

• Specialty formats:

  • Conjugated antibodies (FITC, HRP, Biotin)

  • Carrier-free formulations

How should researchers select the appropriate FAHD2A antibody for their experiments?

Selection of FAHD2A antibodies should be based on:

• Target application: Different antibodies perform optimally in specific applications. For example, some are validated specifically for Western blot, while others work well in immunofluorescence .

• Species reactivity: Most available FAHD2A antibodies react with human FAHD2A, with some showing cross-reactivity with other species:

  • Human-specific antibodies

  • Antibodies with cross-reactivity to monkey

  • Antibodies with cross-reactivity to bovine proteins (92% identity)

• Epitope region: Different antibodies target different regions of the FAHD2A protein:

  • AA 28-77 region antibodies

  • AA 160-279 region antibodies

  • Full-length protein antibodies

• Validation data: Review available validation data including Western blot images, immunofluorescence patterns, and cross-reactivity profiles before selection .

What are the critical considerations for optimizing Western blot protocols with FAHD2A antibodies?

Optimizing Western blot protocols for FAHD2A detection requires attention to several parameters:

• Sample preparation:

  • Use appropriate lysis buffers that preserve protein structure

  • Include protease inhibitors to prevent degradation

  • Heat samples at 95°C for 5 minutes in sample buffer containing SDS and reducing agents

• Antibody selection and dilution:

  • For monoclonal antibodies: typically use 1:2000 dilution

  • For polyclonal antibodies: typically use 1:500-1:2500 dilution

  • Consider using antibodies validated specifically for Western blot applications

• Electrophoresis and transfer conditions:

  • Use 10-12% SDS-PAGE gels for optimal resolution around 31-35 kDa

  • Transfer to PVDF membranes at 100V for 60-90 minutes

• Blocking and detection:

  • Block with 5% non-fat dry milk or BSA in TBST

  • Incubate with primary antibody overnight at 4°C

  • Use HRP-conjugated secondary antibodies with appropriate chemiluminescent detection systems

• Expected results:

  • Primary band at approximately 31-35 kDa

  • Validated with positive controls using FAHD2A-transfected cell lysates

  • Compare with negative controls (empty vector transfected cells)

How can researchers validate FAHD2A antibody specificity and performance?

Comprehensive validation of FAHD2A antibodies should include:

• Positive and negative controls:

  • HEK293T cells transfected with FAHD2A recombinant protein versus empty vector controls

  • Cells with known FAHD2A expression levels (validated by RT-PCR)

  • CRISPR/Cas9 knockout cells as negative controls

• Peptide competition assays:

  • Pre-incubation of antibody with immunizing peptide should abolish specific signals

  • Use synthetic peptides corresponding to the epitope region (e.g., AA 28-77)

• Multiple detection methods:

  • Compare results across different techniques (WB, IF, IHC)

  • Use multiple antibodies targeting different epitopes of FAHD2A

• Signal specificity assessment:

  • Evaluate band pattern in Western blot (single band at expected MW)

  • Assess subcellular localization in IF (expected mitochondrial pattern)

  • Examine tissue expression patterns in IHC that match known distributions

What approaches can be used to investigate FAHD2A protein-protein interactions?

To study FAHD2A protein interactions, researchers can employ several complementary approaches:

• Co-immunoprecipitation (Co-IP):

  • Use FAHD2A antibodies validated for IP applications

  • Perform reverse Co-IP with antibodies against suspected interacting partners

  • Include appropriate controls (IgG, lysate input)

  • Use mild lysis conditions to preserve protein complexes

• Proximity labeling methods:

  • BioID or APEX2 fusion proteins to identify proximal proteins

  • Express FAHD2A fused to promiscuous biotin ligase in cells

  • Purify biotinylated proteins and identify by mass spectrometry

• Fluorescence techniques:

  • FRET or BRET assays with fluorescently-tagged FAHD2A and candidate partners

  • Use FAHD2A antibodies for Proximity Ligation Assay (PLA)

  • Perform co-localization studies using FAHD2A antibodies in combination with antibodies against potential interacting proteins

• Crosslinking mass spectrometry:

  • Chemical crosslinking of protein complexes followed by MS analysis

  • Particularly useful for transient interactions

What are the methodological considerations for using FAHD2A antibodies in immunofluorescence studies?

For optimal immunofluorescence results with FAHD2A antibodies, consider:

• Fixation and permeabilization:

  • For mitochondrial proteins like FAHD2A, paraformaldehyde fixation (4%, 15 min) followed by Triton X-100 permeabilization (0.1%, 10 min) is typically effective

  • Methanol fixation can be used as an alternative that simultaneously fixes and permeabilizes

• Antibody dilution and incubation:

  • Typical dilutions range from 1:100 to 1:500

  • Incubate overnight at 4°C or 1-2 hours at room temperature

  • Include washing steps with PBS containing 0.1% Tween-20

• Controls and counterstaining:

  • Include a mitochondrial marker (MitoTracker, TOM20 antibody) for co-localization

  • Use DAPI for nuclear counterstaining

  • Include secondary antibody-only controls

• Image acquisition:

  • Use confocal microscopy for precise subcellular localization

  • Acquire z-stacks for complete spatial information

  • Use appropriate filter sets to minimize bleed-through

• Expected pattern:

  • Primarily mitochondrial localization with punctate or reticular pattern

  • Validate specificity with FAHD2A-overexpressing and FAHD2A-knockout cells

How do gene editing approaches complement antibody-based FAHD2A research?

Gene editing tools provide powerful complementary approaches to antibody-based FAHD2A research:

• CRISPR/Cas9 knockout systems:

  • FAHD2A CRISPR/Cas9 knockout plasmids are available for human and mouse models

  • Generate complete knockout cells as negative controls for antibody validation

  • Study phenotypic consequences of FAHD2A loss

• CRISPR activation systems:

  • FAHD2A CRISPR activation plasmids can upregulate endogenous FAHD2A expression

  • Useful for studying dose-dependent effects without exogenous protein expression

  • Available in both plasmid and lentiviral formats

• HDR and Double Nickase approaches:

  • FAHD2A HDR (Homology Directed Repair) plasmids allow precise gene editing

  • Double Nickase plasmids reduce off-target effects

  • Enable tagging of endogenous FAHD2A with reporter proteins or epitope tags

• Integration with antibody-based methods:

  • Use edited cells for rigorous antibody validation

  • Combine with immunoprecipitation to study protein complexes under physiological expression levels

  • Create cell lines with tagged FAHD2A for antibody-independent detection

How can researchers address non-specific binding in FAHD2A antibody applications?

Non-specific binding is a common challenge that can be addressed through:

• Optimization of blocking conditions:

  • Test different blocking agents (BSA, non-fat milk, normal serum)

  • Increase blocking time or concentration

  • Include blocking peptides derived from non-specific binding regions

• Antibody dilution adjustment:

  • Titrate antibody concentration to find optimal signal-to-noise ratio

  • Typical Western blot dilutions range from 1:500 to 1:2,500

  • For immunofluorescence, starting at 1:100 is recommended

• Buffer optimization:

  • Add 0.1-0.3% Triton X-100 or 0.05% Tween-20 to reduce hydrophobic interactions

  • Adjust salt concentration (150-500 mM NaCl) to reduce ionic interactions

  • Consider adding 5-10% serum from the secondary antibody host species

• Alternate antibody selection:

  • Try antibodies targeting different epitopes of FAHD2A

  • Compare monoclonal versus polyclonal antibodies

  • Consider using different host species to minimize background

What approaches can optimize FAHD2A detection in challenging tissue samples?

For challenging tissue samples:

• Antigen retrieval optimization:

  • Test both heat-induced epitope retrieval (citrate buffer pH 6.0, EDTA buffer pH 9.0)

  • Adjust retrieval duration (10-30 minutes)

  • Consider enzymatic retrieval for certain fixatives

• Signal amplification strategies:

  • Use tyramide signal amplification (TSA) systems

  • Consider biotin-streptavidin amplification

  • Try polymer-based detection systems

• Tissue-specific considerations:

  • Optimize fixation protocols for specific tissue types

  • Adjust section thickness (4-8 μm typically works well)

  • Block endogenous peroxidase activity in tissues with high peroxidase content

• Controls and validation:

  • Include tissue samples with known FAHD2A expression levels

  • Use peptide competition or FAHD2A knockout controls

  • Compare results from multiple antibodies targeting different epitopes

How can FAHD2A antibodies contribute to mitochondrial research?

FAHD2A antibodies enable several approaches in mitochondrial research:

• Subcellular localization studies:

  • Immunofluorescence co-localization with mitochondrial markers

  • Subcellular fractionation followed by Western blot analysis

  • Immuno-electron microscopy for high-resolution localization

• Mitochondrial stress responses:

  • Monitor FAHD2A expression changes under oxidative stress

  • Assess FAHD2A levels during mitochondrial biogenesis or mitophagy

  • Study FAHD2A in models of mitochondrial disease

• Protein-protein interaction networks:

  • Immunoprecipitation of FAHD2A to identify mitochondrial binding partners

  • Study co-regulation with other mitochondrial proteins

  • Assess FAHD2A incorporation into mitochondrial complexes

• Enzymatic activity correlation:

  • Correlate FAHD2A protein levels with oxaloacetate tautomerase activity

  • Determine relationship between FAHD2A abundance and metabolic flux

What methodological approaches enable studying FAHD2A in metabolic pathways?

To investigate FAHD2A's role in metabolism:

• Metabolic flux analysis:

  • Use FAHD2A antibodies to correlate protein levels with metabolic changes

  • Combine with stable isotope labeling to track carbon flow

  • Compare wild-type and FAHD2A-modulated cells

• Multi-omics integration:

  • Correlate FAHD2A protein levels (using validated antibodies) with:

    • Transcriptomic changes (RNA-seq)

    • Metabolomic alterations (MS or NMR-based metabolomics)

    • Proteomic shifts in relevant pathways

• Enzymatic assays:

  • Immunopurify FAHD2A using specific antibodies for activity assays

  • Correlate enzyme activity with protein levels in various conditions

  • Evaluate post-translational modifications affecting activity

• Tissue-specific metabolism:

  • Use immunohistochemistry to assess FAHD2A distribution across tissues with different metabolic profiles

  • Compare expression patterns with known metabolic enzymes

  • Analyze FAHD2A in metabolic disease models

What emerging techniques will enhance FAHD2A antibody applications?

Emerging technologies that will impact FAHD2A antibody research include:

• Super-resolution microscopy:

  • STORM, PALM, or STED microscopy for nanoscale localization

  • Multi-color super-resolution for co-localization studies

  • Live-cell super-resolution to track FAHD2A dynamics

• Single-cell proteomics:

  • Antibody-based single-cell protein quantification

  • Mass cytometry (CyTOF) for multiplexed protein detection

  • Integration with single-cell transcriptomics

• Advanced multiplexing:

  • Cyclic immunofluorescence for detecting multiple proteins in the same sample

  • Mass spectrometry imaging with antibody-based detection

  • Multiplexed ion beam imaging (MIBI) for spatial proteomics

• In situ proximity labeling:

  • Antibody-enzyme conjugates for spatially-resolved interactome mapping

  • APEX2 or TurboID fusion antibodies for targeted proximity labeling

  • Integration with mass spectrometry for unbiased interaction discovery

How can computational approaches enhance FAHD2A antibody-based research?

Computational methods offer powerful tools to extend FAHD2A antibody applications:

• Machine learning for image analysis:

  • Automated quantification of FAHD2A immunostaining patterns

  • Deep learning for subcellular localization classification

  • Computer vision approaches for co-localization analysis

• Network analysis:

  • Integration of FAHD2A antibody-derived interaction data into protein networks

  • Pathway enrichment analysis of FAHD2A-associated proteins

  • Prediction of functional associations based on co-expression patterns

• Structural biology integration:

  • Mapping antibody epitopes onto predicted protein structures

  • Molecular dynamics simulations to understand antibody-antigen interactions

  • Structure-based prediction of protein-protein interfaces

• Multi-omics data integration:

  • Correlation of FAHD2A protein levels with transcriptomic and metabolomic data

  • System-level modeling of FAHD2A in metabolic networks

  • Causal inference methods to determine FAHD2A's position in regulatory hierarchies