P4H2 Antibody

What is the P4H2 antibody and what does it target?

The P4H2 antibody is a mouse monoclonal antibody specifically designed to target the C-terminus of human DUX4 (Double homeobox 4) protein. It was developed as part of an effort to create reliable tools for detecting DUX4 protein expression in facioscapulohumeral dystrophy (FSHD) research. This antibody has been validated for multiple applications including Western blotting, immunocytochemistry, immunohistochemistry, and ELISA . Unlike some other DUX4 antibodies, P4H2 specifically recognizes DUX4 and does not cross-react with DUX4c, a related protein that shares more than two-thirds of its sequence with DUX4 .

How does P4H2 antibody distinguish between DUX4 and the related DUX4c protein?

The P4H2 antibody was specifically generated against the unique C-terminal region of DUX4 that is not present in DUX4c. This strategic targeting enables researchers to differentiate between these two highly similar proteins that share over two-thirds of their N-terminal sequences. The specificity is crucial because both DUX4 and DUX4c have been proposed as candidate genes for FSHD, making it essential to distinguish their individual contributions to the disease pathology . This specificity has been validated through Western blot analyses showing that P4H2 detects exogenously expressed DUX4 but not DUX4c in transfected cells .

What is the recommended storage and handling protocol for P4H2 antibody?

For optimal performance and longevity of the P4H2 antibody, store it at 4°C for short-term use (typically up to one month). For long-term storage, keep the antibody at -20°C and avoid repeated freeze-thaw cycles that can degrade antibody quality and performance . The antibody is typically supplied in PBS with 0.02% sodium azide as a preservative, at a concentration of 1 mg/mL . When preparing working dilutions, it's advisable to use fresh dilutions for each experiment rather than storing diluted antibody for extended periods.

What are the validated applications and optimal dilutions for P4H2 antibody?

The P4H2 antibody has been validated for multiple applications with specific recommended dilutions for each technique:

These recommendations provide starting points that may require optimization based on specific experimental conditions, sample types, and detection methods .

How should samples be prepared for optimal DUX4 detection using P4H2 antibody in immunofluorescence?

For optimal detection of DUX4 in immunofluorescence applications using the P4H2 antibody, cells should be gently washed in PBS and fixed with 2% paraformaldehyde for 7 minutes at room temperature, followed by two PBS washes. Permeabilization should be performed with 1% Triton X-100 in PBS for 10 minutes at room temperature with gentle rocking. For primary antibody incubation, dilute P4H2 at 1:100-1:200 in PBS and incubate overnight at 4°C, followed by PBS washes. Use appropriate fluorophore-conjugated secondary antibodies (such as TRITC or FITC-conjugated anti-mouse IgG) with incubation for 1 hour at room temperature. Counterstain nuclei with DAPI before examining under a fluorescence microscope . This protocol has been validated for detecting nuclear DUX4 in transfected cells and in rare DUX4-expressing cells from FSHD patient samples.

What protocol is recommended for Western blot analysis using P4H2 antibody?

For Western blot analysis with P4H2 antibody, cells should be directly lysed in 2× Laemmli sample buffer and sonicated to shear genomic DNA. Load equal amounts of protein on 10% bis-tris polyacrylamide gels for electrophoresis. Transfer proteins to nitrocellulose membranes and block with 5% non-fat dry milk in PBS containing 0.1% Tween-20 (PBST). Incubate the membrane with P4H2 antibody at 0.5-2.0 μg/mL in PBST overnight at 4°C. After washing with PBST, incubate with HRP-conjugated goat anti-mouse IgG secondary antibody. Develop using an ECL substrate and expose to film . The expected molecular weight of DUX4 is approximately 52 kDa, though the observed molecular weight may vary due to post-translational modifications. For negative controls, include lysates from untransfected cells, and for loading controls, consider reprobing with anti-α-tubulin antibody after stripping the membrane .

How can P4H2 antibody be used to detect endogenous DUX4 expression in FSHD muscle cells?

Detecting endogenous DUX4 in FSHD muscle cells is challenging due to its low expression levels and restricted expression to a small percentage of cells. For immunocytochemistry detection of endogenous DUX4, differentiated myogenic cells from FSHD patients should be fixed with 2% paraformaldehyde and permeabilized with 1% Triton X-100. Incubate with P4H2 antibody at 1:100 dilution overnight at 4°C, followed by fluorophore-conjugated secondary antibody. Nuclear staining in approximately 0.1% of cultured FSHD muscle cells is expected . For enhanced sensitivity, consider combining immunofluorescence with other techniques like RT-PCR or using enrichment strategies for DUX4-expressing cells. The antibody has been validated to detect full-length DUX4 (DUX4-FL) nuclear immunoreactivity at a higher frequency among differentiated CD56+ myogenic cells from FSHD compared to non-FSHD individuals .

What are the expected staining patterns for DUX4 using P4H2 antibody and how do they differ between FSHD and control samples?

When using P4H2 antibody, DUX4 staining shows a distinct nuclear localization pattern, consistent with its function as a transcription factor. In cells expressing exogenous DUX4 (such as transfected C2C12 myoblasts), strong nuclear staining is observed. In FSHD patient-derived muscle cells, DUX4 expression is detected in approximately 0.1% of nuclei . The staining pattern in DUX4-positive nuclei from FSHD samples often exhibits a punctate, granular appearance suggestive of chromatin condensation, potentially indicating early stages of apoptosis . This pattern may reflect DUX4's role in triggering cell death pathways. In contrast, control samples from unaffected individuals typically show no detectable DUX4 staining in differentiated muscle cells. When comparing FSHD and control samples, researchers should examine multiple fields to account for the rare expression pattern and consider co-staining with markers of cell death to correlate DUX4 expression with cellular pathology.

How can P4H2 antibody be used alongside other DUX4 antibodies in co-localization studies?

P4H2 antibody (mouse monoclonal) can be effectively paired with antibodies raised in different species, such as rabbit monoclonal antibodies targeting different epitopes of DUX4, for co-localization studies. For instance, P4H2 (targeting the C-terminus) can be used in combination with E14-3 (a rabbit monoclonal targeting the N-terminus of DUX4) to confirm the presence of full-length DUX4 . For co-staining protocols, fix and permeabilize cells as described previously, then incubate simultaneously with both primary antibodies at their optimal dilutions (typically 1:100-1:200). After washing, incubate with species-specific secondary antibodies conjugated to different fluorophores (e.g., FITC-conjugated anti-mouse for P4H2 and TRITC-conjugated anti-rabbit for E14-3). This approach allows visualization of multiple epitopes simultaneously and can provide stronger evidence for the presence of intact DUX4 protein, particularly in samples with low expression levels .

How can P4H2 antibody be used to study the differential effects of DUX4 isoforms in cellular models?

The P4H2 antibody can be instrumental in differentiating between the effects of distinct DUX4 isoforms in cellular models. Since P4H2 targets the C-terminus of DUX4, it specifically detects the full-length DUX4 (DUX4-fl) but not the shorter splice variant (DUX4-s) that lacks the C-terminal domain. This specificity allows researchers to distinguish between these isoforms, which is crucial as they have profoundly different biological effects. In experimental designs, researchers can use lentiviral constructs expressing either full-length or short DUX4 and detect their expression patterns using P4H2 for DUX4-fl and N-terminal targeting antibodies for both isoforms . Studies have shown that while full-length DUX4 induces cell death in human primary muscle cells, the shorter splice form shows no such toxicity. By immunostaining with P4H2, researchers can correlate DUX4-fl expression with cellular phenotypes like nuclear condensation, activation of caspase pathways, and altered gene expression profiles to elucidate the mechanisms of DUX4-mediated pathology in FSHD .

What strategies can be employed to improve detection sensitivity for rare DUX4-expressing cells in FSHD samples?

Detecting the rare DUX4-expressing cells in FSHD samples requires optimized strategies to enhance sensitivity:

• Signal Amplification Methods: Use tyramide signal amplification (TSA) or similar methods to enhance fluorescence signal while maintaining specificity.

• Multi-antibody Approach: Combine P4H2 with other validated DUX4 antibodies targeting different epitopes, using distinct fluorophores for co-localization studies.

• Enrichment of DUX4-positive Cells: Consider using FACS-based methods after staining for DUX4 target genes or reporters to enrich for cells likely to express DUX4.

• Optimized Fixation and Permeabilization: Test alternative fixation methods such as methanol fixation or different concentrations of paraformaldehyde (1-4%) and permeabilization reagents.

• Enhanced Imaging Techniques: Utilize high-resolution confocal microscopy or super-resolution microscopy techniques to detect faint or localized signals.

• Automated High-throughput Screening: Implement automated imaging platforms to scan large numbers of cells to identify the rare positive cells (typically ~0.1% of FSHD muscle cells) .

These approaches, particularly when used in combination, can significantly improve the detection of DUX4-expressing cells in FSHD patient samples, facilitating more robust data collection and analysis.

How can P4H2 antibody be used in chromatin immunoprecipitation (ChIP) experiments to study DUX4 binding sites?

While not explicitly demonstrated in the provided search results, P4H2 antibody can theoretically be adapted for chromatin immunoprecipitation (ChIP) experiments to investigate DUX4 binding sites genome-wide. For a ChIP protocol using P4H2 antibody, researchers should first optimize crosslinking conditions (typically 1% formaldehyde for 10 minutes) for muscle cells or appropriate cell models expressing DUX4. After cell lysis and sonication to generate chromatin fragments of approximately 200-500 bp, immunoprecipitation can be performed using 2-5 μg of P4H2 antibody per reaction, with appropriate pre-clearing steps and IgG controls. Due to the low expression of DUX4 in FSHD cells, consider using cell models with inducible or stable DUX4 expression for initial optimization. Alternatively, for endogenous studies, sequential ChIP (re-ChIP) approaches combining P4H2 with antibodies to known DUX4-associated factors might enhance specificity. The precipitated DNA can be analyzed by qPCR for known DUX4 targets or by sequencing (ChIP-seq) for genome-wide binding site identification. Since DUX4 contains homeodomains that bind to specific DNA sequences, correlation of ChIP data with gene expression changes can provide insights into direct transcriptional targets of DUX4 that contribute to FSHD pathology.

What are common issues encountered when using P4H2 antibody and how can they be resolved?

When working with P4H2 antibody, researchers may encounter several common issues that can affect experimental outcomes:

When troubleshooting, always include positive controls (cells transfected with DUX4) and negative controls (untransfected cells or non-relevant antibody of the same isotype) .

How should researchers validate the specificity of P4H2 antibody in their experimental systems?

To validate the specificity of P4H2 antibody in new experimental systems, researchers should implement a comprehensive validation strategy:

• Positive and Negative Controls: Include cells transfected with DUX4 as positive controls and untransfected cells as negative controls. Additionally, cells expressing DUX4c but not DUX4 can demonstrate the antibody's specificity for DUX4 over DUX4c .

• Peptide Competition Assay: Pre-incubate the antibody with the immunizing peptide (C-terminal DUX4 peptide) before application to samples. This should abolish specific staining.

• Knockdown/Knockout Validation: Use DUX4 siRNA or CRISPR/Cas9 to reduce or eliminate DUX4 expression in positive control samples, confirming that signal disappears accordingly.

• Multiple Antibody Comparison: Compare results with other validated DUX4 antibodies targeting different epitopes (e.g., comparing P4H2 with E14-3 or 9A12) .

• Correlation with mRNA Expression: Combine protein detection with RT-PCR for DUX4 mRNA to confirm concordance between protein and transcript detection.

• Cross-species Reactivity Testing: The P4H2 antibody is designed for human DUX4 detection. If using in other species, expression of human DUX4 constructs can serve as positive controls to validate cross-reactivity.

These validation steps ensure that experimental results accurately reflect DUX4 expression patterns and minimize the risk of misinterpretation due to antibody cross-reactivity or non-specific binding.

How can researchers distinguish between specific and non-specific signals when detecting low-abundance DUX4 in FSHD samples?

Distinguishing specific from non-specific signals when detecting low-abundance DUX4 in FSHD samples requires rigorous experimental design and controls:

• Multiple Antibody Validation: Confirm positive signals using at least two different antibodies targeting distinct epitopes of DUX4 (e.g., combining P4H2 with E14-3 or other validated antibodies) .

• Characteristic Staining Pattern: Authentic DUX4 signal should show nuclear localization with the punctate, granular pattern described in validated studies .

• Correlation with DUX4 Target Genes: Complement protein detection with analysis of known DUX4 target genes by RT-PCR or RNA-FISH in the same samples.

• Inclusion of Multiple Controls:

  • Technical negative controls (secondary antibody only)

  • Biological negative controls (muscle cells from unaffected individuals)

  • Isotype controls (irrelevant antibody of same isotype and concentration)

  • Positive controls (cells transfected with DUX4)

• Signal Quantification: Implement objective quantification methods to distinguish real signals from background, such as signal-to-noise ratio calculations.

• Sequential Staining Protocol: For critical samples, consider a sequential staining approach where samples are first imaged after secondary antibody addition, then stained with antibodies to known DUX4 target proteins to confirm correlation.

• Single-Cell Analysis: When possible, correlate DUX4 immunostaining with single-cell RNA-seq or other single-cell analyses to confirm the biological signature of DUX4-expressing cells.

These approaches collectively increase confidence in distinguishing genuine DUX4 expression from background or artifactual signals in FSHD samples, where the true signal may be present in only approximately 0.1% of muscle nuclei .

How can P4H2 antibody be integrated into high-throughput screening assays for DUX4 inhibitors?

The P4H2 antibody can be effectively integrated into high-throughput screening (HTS) platforms to identify potential DUX4 inhibitors for FSHD therapeutics. A methodological approach includes:

• Assay Development: Establish a cell-based system with inducible or stable DUX4 expression that can be detected by P4H2 antibody. C2C12 myoblasts or immortalized FSHD myoblasts are suitable model systems .

• Automation-Compatible Format: Adapt immunofluorescence protocols for 96- or 384-well plate formats with automated liquid handling and imaging systems.

• Quantitative Readouts: Develop robust quantification methods for:

  • DUX4 protein levels (nuclear P4H2 staining intensity)

  • Percentage of DUX4-positive nuclei

  • Nuclear morphology changes associated with DUX4 expression

  • Cell viability parameters

• Compound Testing Workflow:

  • Treat cells with compound libraries

  • Fix and immunostain using P4H2 antibody (1:200 dilution)

  • Counterstain nuclei with DAPI

  • Image using automated microscopy

  • Analyze using machine learning algorithms for multiparametric phenotyping

• Validation Strategy: Confirm hits using orthogonal assays:

  • Western blot verification of DUX4 reduction

  • RT-PCR analysis of DUX4 target genes

  • Secondary assays for functional outcomes (cell survival, differentiation capacity)

This approach leverages the specificity of P4H2 antibody to enable large-scale screening for compounds that reduce DUX4 protein expression or mitigate its downstream effects, potentially accelerating therapeutic discovery for FSHD.

What are the potential applications of P4H2 antibody in examining the role of DUX4 in embryonic development and cancer?

Beyond FSHD research, the P4H2 antibody has potential applications in studying DUX4's physiological and pathological roles in other contexts:

• Embryonic Development Studies:

  • DUX4 has been implicated in early embryonic gene activation and cleavage-stage embryo development

  • P4H2 antibody can be used to detect endogenous DUX4 expression in human embryonic tissues

  • Immunohistochemistry on embryonic samples can map the spatiotemporal expression pattern of DUX4

  • Co-localization studies with developmental markers can elucidate DUX4's role in lineage specification

• Cancer Research Applications:

  • DUX4 reactivation has been reported in certain cancers, including subsets of leukemia with DUX4 rearrangements

  • Tissue microarray screening with P4H2 antibody can identify tumors with aberrant DUX4 expression

  • Correlation studies between DUX4 expression and patient outcomes may identify prognostic biomarkers

  • Mechanistic studies examining DUX4's interaction with oncogenic pathways could identify therapeutic vulnerabilities

• Comparative Studies:

  • Analysis of DUX4 expression in germline tissues (where it may have physiological roles) versus pathological expression in FSHD and cancer

  • Comparison of post-translational modifications of DUX4 in different cellular contexts using immunoprecipitation with P4H2 followed by mass spectrometry

These applications extend the utility of P4H2 antibody beyond FSHD research into fundamental biological questions regarding DUX4's diverse roles in development and disease.

How can advanced imaging techniques enhance the utility of P4H2 antibody for studying DUX4 dynamics in living cells?

While standard applications of P4H2 involve fixed cell immunostaining, adapting the antibody for advanced live-cell imaging techniques could provide unprecedented insights into DUX4 dynamics:

• Derivation of scFv or Fab Fragments:

  • Engineer single-chain variable fragments (scFv) or Fab fragments from P4H2 that retain DUX4 binding specificity

  • These smaller antibody derivatives can be expressed intracellularly or loaded into cells for live-cell applications

• Fluorescent Protein Fusions for Live Imaging:

  • Express scFv-GFP fusions intracellularly to track endogenous DUX4

  • Combine with photoactivatable or photoswitchable fluorescent proteins for pulse-chase experiments tracking DUX4 turnover

• Super-Resolution Microscopy Applications:

  • Conjugate P4H2 with appropriate fluorophores for STORM, PALM, or STED microscopy

  • Examine nanoscale organization of DUX4 in nuclear compartments with 10-20 nm resolution

  • Perform co-localization studies with chromatin markers at super-resolution level

• Correlative Light and Electron Microscopy (CLEM):

  • Use P4H2 for fluorescence imaging followed by electron microscopy of the same sample

  • Examine ultrastructural features of DUX4-expressing nuclei at nanometer resolution

• Lattice Light-Sheet Microscopy:

  • Apply to DUX4-inducible systems with fluorescently tagged P4H2 derivatives

  • Capture rapid 3D dynamics of DUX4 expression and localization with minimal phototoxicity

• Single-Molecule Tracking:

  • Label P4H2 Fab fragments with quantum dots or organic dyes

  • Track individual DUX4 molecules to determine diffusion coefficients, binding kinetics, and interactions with chromatin

These advanced imaging approaches would transform P4H2 from a static detection tool into a dynamic probe for understanding DUX4 behavior in living systems, potentially revealing new aspects of DUX4 biology relevant to FSHD pathogenesis.