PDHX Antibody

What is PDHX and why is it important in metabolic research?

PDHX functions as the E3-binding protein (E3BP) within the pyruvate dehydrogenase complex (PDC). It is critical for anchoring dihydrolipoamide dehydrogenase (E3) to the dihydrolipoamide transacetylase (E2) core of the pyruvate dehydrogenase complexes in eukaryotes. This specific binding is essential for a functional PDH complex, which plays a central role in cellular energy metabolism . PDHX is predominantly expressed in tissues with high energy demands such as the heart and skeletal muscle, making it particularly relevant for metabolic and cardiac research . Understanding PDHX function is critical for researchers studying metabolic disorders, energy metabolism pathways, and mitochondrial function.

What species reactivity can be expected from commercially available PDHX antibodies?

Most commercially available PDHX antibodies demonstrate reactivity to human, mouse, and rat PDHX proteins, making them versatile tools for comparative studies across these common laboratory model systems . Some antibodies, like the one from Abcam (ab232798), also show reactivity with pig samples . When selecting a PDHX antibody, researchers should verify the specific species reactivity claims and whether these have been experimentally validated or are predicted based on sequence homology. Cross-reactivity with additional species may exist but would typically require experimental validation before use in critical experiments.

What applications are PDHX antibodies commonly validated for?

PDHX antibodies are typically validated for multiple applications, with the most common being:

Researchers should consider that performance may vary between applications and should conduct preliminary validation experiments to determine optimal conditions for their specific experimental systems .

How do different immunogens affect the specificity of PDHX antibodies?

Different vendors use varying immunogen strategies for PDHX antibody production, which can impact epitope recognition and specificity:

• Abcam's rabbit polyclonal antibody (ab232798) uses a recombinant fragment within human PDHX aa 200-500

• Abbexa's antibody utilizes a recombinant fusion protein corresponding to amino acids 1-300 of human PDHX

• Prospec Bio's monoclonal antibody is derived from mice immunized with recombinant human PDHX amino acids 54-501

• Boster Bio's antibody uses E.coli-derived human PDHX recombinant protein position M1-L500

• R&D Systems' antibody is raised against E. coli-derived recombinant human PDHX aa 387-501

These differences in immunogen design can affect epitope recognition, potentially leading to variability in antibody performance across different experimental conditions or when detecting specific PDHX isoforms. When studying specific domains or post-translational modifications of PDHX, researchers should select antibodies with immunogens encompassing the region of interest and conduct validation experiments to confirm appropriate epitope recognition.

What are the optimal storage conditions for maintaining PDHX antibody activity?

To preserve antibody activity, most PDHX antibodies should be:

• Stored at -20°C for long-term storage (typically 12 months from receipt)

• Aliquoted upon first thawing to minimize freeze-thaw cycles

• For short-term storage (up to 1-6 months after reconstitution), some antibodies can be stored at 2-8°C

Most commercially available PDHX antibodies are supplied in formulations containing stabilizers such as glycerol (typically 50%), which prevents freezing at -20°C and maintains antibody stability . Sodium azide (0.02%) is commonly included as a preservative . Repeated freeze-thaw cycles should be strictly avoided as they can lead to protein denaturation and loss of antibody activity . If working with a lyophilized antibody, reconstitution should follow manufacturer specifications precisely to ensure optimal performance.

What controls should be included when validating a PDHX antibody for research use?

A comprehensive validation strategy for PDHX antibodies should include:

• Positive Controls:

  • Recombinant human PDHX protein

  • Tissue lysates known to express high levels of PDHX (heart and skeletal muscle)

  • Cell lines with confirmed PDHX expression (such as 293T cells)

• Negative Controls:

  • Antibody diluent only (no primary antibody)

  • Pre-incubation of antibody with immunizing peptide/protein (peptide blocking)

  • Tissues or cells with PDHX knocked down or knocked out (if available)

• Loading Controls:

  • For Western blot, include housekeeping proteins (β-actin, GAPDH) to normalize expression levels

  • For IHC/ICC, include serial sections with isotype control antibodies

These controls help distinguish specific from non-specific signals and provide confidence in antibody specificity. When comparing results across different studies or antibody lots, researchers should conduct side-by-side comparisons with previously validated antibodies to ensure consistency in detection patterns.

How should I optimize PDHX antibody dilutions for Western blot applications?

Optimizing PDHX antibody dilutions for Western blot requires systematic testing:

• Begin with the manufacturer's recommended dilution range (typically 1:500-1:2000 for many PDHX antibodies)

• Perform a dilution series experiment:

  • Test 3-4 dilutions spanning the recommended range

  • Include a positive control sample (e.g., heart tissue lysate)

  • Process all blots identically (same blocking, washing, and detection conditions)

• Assess results based on:

  • Signal-to-noise ratio

  • Specificity (single band at expected 54 kDa)

  • Background levels

  • Signal intensity relative to loading controls

• For quantitative Western blots:

  • Verify the antibody produces a linear response across a range of protein concentrations

  • Determine the dynamic range where signal intensity correlates with protein amount

Remember that optimal dilutions may vary depending on the detection system used (chemiluminescence, fluorescence, or colorimetric) and sample type. Additionally, antibody lots may show some variation, so optimization may need to be repeated when using a new lot.

What are the recommended protocols for PDHX detection in immunohistochemistry?

For optimal PDHX detection in immunohistochemistry, follow these methodological guidelines:

• Tissue Preparation and Fixation:

  • Formalin-fixed paraffin-embedded (FFPE) tissues are suitable for PDHX detection

  • Optimal fixation time (typically 24 hours) balances epitope preservation and tissue morphology

• Antigen Retrieval:

  • Heat-induced epitope retrieval (HIER) is typically required

  • Try citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) to determine optimal conditions

• Blocking and Antibody Incubation:

  • Block with 5-10% normal serum from the same species as the secondary antibody

  • Use 1:50-1:400 dilution of primary PDHX antibody

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

• Detection System:

  • Use a detection system compatible with the host species of the primary antibody

  • For polyclonal rabbit PDHX antibodies, anti-rabbit HRP-polymer systems work well

  • Include 3,3'-diaminobenzidine (DAB) exposure controls to ensure consistent development

• Controls and Interpretation:

  • Include positive tissue controls (heart, skeletal muscle)

  • Assess both staining intensity and subcellular localization

  • PDHX should show predominantly mitochondrial localization

This protocol should be optimized for each specific antibody and tissue type being studied. Particular attention should be paid to antigen retrieval conditions, as these often significantly impact staining quality for mitochondrial proteins like PDHX.

How can I accurately quantify PDHX expression levels in Western blot analysis?

For accurate quantification of PDHX expression:

• Optimize Protein Loading:

  • Determine the linear range of detection for your specific antibody and system

  • Use 10-30 μg of total protein per lane for most cell/tissue lysates

  • Include a standard curve of recombinant PDHX if absolute quantification is needed

• Image Acquisition:

  • Capture images before signal saturation occurs

  • Use a digital imaging system with a wide dynamic range

  • For chemiluminescence, capture multiple exposure times

• Normalization Strategy:

  • Normalize PDHX signal to housekeeping proteins (β-actin, GAPDH)

  • Consider using total protein normalization (Ponceau S or Stain-Free gels) for more accurate results

  • For tissues with variable mitochondrial content, consider normalizing to other mitochondrial proteins

• Analysis Software:

  • Use software that can perform density analysis (ImageJ, Image Lab, etc.)

  • Subtract background signal from each lane

  • Compare band intensities only within the linear range of detection

• Statistical Analysis:

  • Perform experiments in biological triplicates at minimum

  • Use appropriate statistical tests to determine significance

  • Report both raw and normalized data

This approach helps control for technical variations and ensures that observed differences in PDHX expression accurately reflect biological reality rather than experimental artifacts.

What factors might cause non-specific binding with PDHX antibodies and how can these be minimized?

Several factors can contribute to non-specific binding with PDHX antibodies:

• Antibody-Related Factors:

  • Polyclonal antibodies may contain antibodies against contaminants in the immunogen

  • High antibody concentrations can increase non-specific binding

  • Solution: Use affinity-purified antibodies at optimized concentrations

• Sample Preparation Issues:

  • Incomplete protein denaturation can cause aggregation and non-specific binding

  • Insufficient blocking allows primary antibody to bind to the membrane

  • Solution: Ensure complete denaturation with appropriate buffers and optimize blocking conditions

• Cross-Reactivity:

  • Antibodies may recognize epitopes shared between PDHX and other proteins

  • Solution: Verify antibody specificity using knockout/knockdown controls or peptide blocking experiments

• Protocol Optimization:

  • Insufficient washing can leave residual antibody causing background

  • Inappropriate blocking agents may not block all non-specific sites

  • Solution: Increase washing time/stringency and optimize blocking conditions

• Specific Recommendations for PDHX:

  • Use 5% non-fat dry milk or 3-5% BSA in TBST for blocking

  • Consider using specialized immunoblot buffers (e.g., "Immunoblot Buffer Group 1" has been validated for some PDHX antibodies)

  • For high background in Western blots, try reducing primary antibody concentration and extending incubation time (overnight at 4°C)

By systematically addressing these factors, researchers can significantly improve the specificity of PDHX detection in their experiments.

How should I interpret discrepancies in PDHX detection between different antibody clones?

When facing discrepancies between different PDHX antibody clones:

• Consider Epitope Differences:

  • Different antibodies recognize distinct epitopes within the PDHX protein

  • Monoclonal antibodies (like Prospec Bio's) recognize single epitopes, while polyclonal antibodies (like Abcam's) recognize multiple epitopes

  • Compare the immunogen sequences used to generate each antibody

• Evaluate Post-translational Modifications:

  • Some antibodies may have differential sensitivity to phosphorylated PDHX (known phosphorylation sites include Ser75 and Ser130)

  • Modifications may mask epitopes recognized by specific antibodies

• Assess Isoform Specificity:

  • Human PDHX has multiple secondary UniProt accession numbers (B4DW62, D3DR11, E9PB14, E9PBP7, O60221, Q96FV8, Q99783) , suggesting potential isoforms

  • Different antibodies may have varying affinities for specific isoforms

• Protocol-Dependent Variables:

  • Detection methods (chemiluminescence vs. fluorescence)

  • Sample preparation conditions (reducing vs. non-reducing)

  • Buffer compositions may affect epitope accessibility

• Resolution Strategies:

  • Use multiple antibodies targeting different regions of PDHX

  • Validate with orthogonal techniques (mass spectrometry, RNA expression)

  • Consider the biological context and expected expression pattern

Understanding the basis for these discrepancies is crucial for proper data interpretation and experimental design, particularly in studies exploring PDHX regulation and function under different physiological or pathological conditions.

What are the considerations for using PDHX antibodies in co-immunoprecipitation studies?

When designing co-immunoprecipitation (co-IP) experiments to study PDHX interactions:

• Antibody Selection:

  • Choose antibodies specifically validated for IP applications

  • Consider using antibodies targeting different epitopes than those in detection antibodies

  • For IP applications, use 0.5-4 μg antibody per 400-600 μg of cell/tissue extract

• Lysis Conditions:

  • Use non-denaturing lysis buffers to preserve protein-protein interactions

  • For mitochondrial proteins like PDHX, ensure mitochondrial membranes are properly solubilized

  • Consider specialized mitochondrial isolation protocols prior to co-IP

• Experimental Controls:

  • Include IgG isotype control to identify non-specific binding

  • Use lysates from cells with PDHX knockdown as negative controls

  • Perform reverse IP (immunoprecipitate with antibodies against suspected interaction partners)

• Detection Strategy:

  • For Western blot detection after IP, use antibodies from different host species to avoid detecting the IP antibody

  • Consider native elution conditions to preserve complexes for further analysis

• Known Interactions to Validate:

  • PDHX interactions with E3 (dihydrolipoamide dehydrogenase) and E2 (dihydrolipoamide transacetylase) components of the PDH complex

  • These validated interactions can serve as positive controls

Co-IP experiments are particularly valuable for studying how PDHX tethers E3 dimers to the E2 core of the pyruvate dehydrogenase complex and how this interaction may be regulated under different physiological conditions.

How can I study PDHX expression patterns across different tissue types?

For comprehensive analysis of PDHX expression across tissues:

• Sampling Strategy:

  • Include tissues with expected high expression (heart, skeletal muscle)

  • Include tissues with moderate and low expression for comparison

  • Consider both normal and diseased tissues if studying pathological conditions

• Methodological Approach:

  • Use a multi-platform approach combining:

    • Western blot for protein level quantification

    • IHC for spatial distribution within tissues

    • qRT-PCR for mRNA expression correlation

• Normalization Considerations:

  • For Western blot: normalize to total protein rather than single housekeeping genes

  • For IHC: use digital image analysis with standardized acquisition parameters

  • For tissues with varying mitochondrial content: consider dual staining with mitochondrial markers

• Data Interpretation:

  • Create expression heatmaps across tissues

  • Correlate PDHX expression with tissue metabolic activity

  • Compare protein vs. mRNA expression patterns to identify post-transcriptional regulation

• Specialized Techniques:

  • Consider laser capture microdissection for analyzing specific cell populations within heterogeneous tissues

  • Single-cell analysis for cell-type specific expression patterns

  • Proximity ligation assay to study in situ interactions with other PDH complex components

This comprehensive approach provides insights into the tissue-specific regulation of PDHX and its correlation with metabolic demands across different tissues and cell types.

What techniques can confirm PDHX antibody specificity in knockout/knockdown models?

To rigorously validate PDHX antibody specificity:

• Genetic Modification Approaches:

  • CRISPR/Cas9-mediated PDHX knockout cell lines

  • siRNA or shRNA-mediated PDHX knockdown

  • Heterologous expression systems (overexpression of tagged PDHX)

• Validation Techniques:

  • Western blot comparison between wild-type and knockout/knockdown samples

  • Immunocytochemistry with side-by-side comparison

  • Flow cytometry for quantitative analysis of signal reduction

• Experimental Design:

  • Include partial knockdowns to assess antibody sensitivity

  • Test multiple antibody dilutions to determine dynamic range

  • Include rescue experiments (re-expression of PDHX in knockout cells)

• Controls and Interpretation:

  • Verify knockdown/knockout efficiency with qRT-PCR

  • Compare results across multiple PDHX antibodies targeting different epitopes

  • Quantify signal reduction proportional to knockdown efficiency

• Advanced Confirmation Methods:

  • Mass spectrometry to confirm absence of PDHX in immunoprecipitates from knockout cells

  • Peptide competition assays using the immunizing peptide

  • Super-resolution microscopy to confirm loss of mitochondrial localization