DUX4 Antibody, Biotin conjugated

What is DUX4 and why is it a significant research target?

DUX4 (Double Homeobox 4) is a transcription factor that has emerged as a critical research target due to its multifunctional roles in both development and disease. DUX4 features a highly unusual charge distribution, with the N-terminal region having a net charge of +17 and the C-terminal region having a net charge of -15 elementary charge units, placing it at the extremes of the human proteome in terms of charge polarization .

DUX4 is expressed at early pre-implantation stages in human embryos and plays a crucial role in embryonic genome activation (EGA). It activates thousands of transcribed enhancer-like regions, particularly those within ERVL-MaLR repeat elements, contributing to the regulation of embryonic development .

Aberrant expression of DUX4 is associated with multiple diseases, including facio-scapulo-humeral muscular dystrophy (FSHD), acute lymphoblastic leukemia, and various sarcomas, making it an important therapeutic target . The protein's normal molecular weight is approximately 45 kDa .

What are the key specifications of the DUX4 Antibody, Biotin conjugated?

The DUX4 Antibody, Biotin conjugated (such as ABIN2482296) is a monoclonal antibody derived from mouse that specifically targets the C-terminal region of human DUX4. Its key specifications include:

• Binding Specificity: C-Terminal region of DUX4

• Reactivity: Primary reactivity with Human DUX4

• Host: Mouse

• Clonality: Monoclonal (Clone P2B1)

• Isotype: IgG1

• Conjugate: Biotin

• Specificity: Detects ~45 kDa band with no cross-reactivity with DUX4c

• Cross-Reactivity: Human, Mouse

• Purification Method: Protein G Purified

• Immunogen: C-terminal 76 amino acids of DUX4 with glutathione-s-transferase (gst) tag

The biotin conjugation specifically enhances detection sensitivity through avidin/streptavidin-based systems while preserving the antibody's binding characteristics to the target protein.

What applications are compatible with DUX4 Antibody, Biotin conjugated?

The DUX4 Antibody, Biotin conjugated is validated for multiple research applications:

• Western Blotting (WB): Enables detection of denatured DUX4 protein at approximately 45 kDa. The biotin conjugation allows for signal amplification using streptavidin-based detection systems, enhancing sensitivity for low-abundance targets .

• Immunohistochemistry (IHC): Permits visualization of DUX4 localization in tissue sections, particularly useful for studying muscle biopsies in FSHD research or embryonic tissues .

• Immunofluorescence (IF): Enables subcellular localization studies of DUX4, particularly important for nuclear localization assessment during embryonic genome activation where intense nuclear DUX4 localization precedes minor EGA .

• Immunocytochemistry (ICC): Allows detection of DUX4 in cultured cells, particularly useful for studying transfected cell lines expressing either full DUX4 or truncated versions like DUX4_ΔC .

How can researchers optimize DUX4 antibody-based co-immunoprecipitation experiments?

When designing co-immunoprecipitation experiments with DUX4 Antibody, Biotin conjugated, researchers must account for DUX4's unique characteristics:

• Consider charge distribution: DUX4's extreme charge polarization (+17 at N-terminal, -15 at C-terminal) creates challenges for protein interaction studies. Use buffers that minimize nonspecific electrostatic interactions while maintaining physiologically relevant binding partners .

• Validated experimental approach:

  • Transfect cells with plasmids expressing DUX4 (full length) and DUX4_ΔC (C-terminal deletion) as a negative control

  • Allow 48 hours for expression (based on established protocols showing DUX4-induced gene expression starting at 24 hours)

  • Perform cell lysis under conditions that preserve protein-protein interactions

  • Use Streptactin resin for affinity purification of biotin-conjugated antibody complexes

  • Perform mass spectrometry analysis on bound proteins

• Expected interactors: This approach has successfully identified several key DUX4 interactors, including:

  • Histone acetyltransferases p300 and CREBBP (highly enriched in DUX4 pulldown)

  • H3.X and H3.Y histone variants

  • PTOV1 (contains a homologous PTOV-domain like MED25)

  • Components of the Mediator complex through MED25 interaction

  • The entire AP-2 (adaptor protein 2) complex (subunits sigma, mu, gamma, alpha-1 and alpha-2)

What methodologies are effective for studying DUX4's role in embryonic genome activation using biotin-conjugated antibodies?

To investigate DUX4's function in embryonic genome activation, researchers can implement these methodological approaches:

• Zygote analysis: DUX4 antibody staining reveals marked enrichment in human zygotes with intense nuclear localization preceding and coinciding with minor embryonic genome activation .

• Transcribed enhancer activation studies:

  • CRISPR activation of transcribed enhancers by C-terminal DUX4 motifs can be used to study increased expression of target EGA genes like ZSCAN4 and KHDC1P1

  • The biotin-conjugated antibody can validate DUX4 localization at these enhancer regions

• Knockdown validation: Researchers have successfully employed DUX4 knockdown in human zygotes to assess changes in the EGA transcriptome

  • This approach revealed that while DUX4 influences the EGA transcriptome, its knockdown did not terminate embryo development

• Chromatin accessibility studies: Combine antibody-based chromatin immunoprecipitation with ATAC-seq to identify loci associated with DUX4 expression, revealing substantial changes in chromatin accessibility of non-coding DNA and activation of enhancer-like regions .

How can the DUX4 Antibody, Biotin conjugated be used alongside aptamer-based approaches?

Integrating antibody and aptamer approaches creates powerful complementary methodologies:

• Selection of high-affinity aptamers: Researchers have identified DNA aptamers against DUX4 with specific secondary structural elements (hairpins with bulge loops) that confer high affinity for DUX4 protein. These aptamers can be used alongside antibodies for validation .

• Combined detection methodology:

  • Use biotin-conjugated DUX4 antibody for initial protein detection/localization

  • Apply fluorescence resonance energy transfer (FRET) techniques with aptamers to confirm binding and measure affinities

  • Validate interactions using fluorescence polarization techniques

• Structure-function relationship studies: Crystal structure analysis of DUX4-aptamer complexes revealed that aptamers contain two consensus DUX4 motifs in reverse complementary fashion, forming hairpins with bulge loops that enlarge the binding surface with DUX4 protein .

What are common technical challenges when using DUX4 Antibody, Biotin conjugated for Western blotting?

When performing Western blot analysis with DUX4 Antibody, Biotin conjugated, researchers should address these common challenges:

• Background issues: The biotin conjugation may increase background in tissues with high endogenous biotin. Implement blocking steps with avidin/biotin blocking reagents before applying the primary antibody.

• Signal specificity:

  • Expected molecular weight: ~45 kDa band for full-length DUX4

  • No cross-reactivity with DUX4c should be observed

  • Validate specificity using DUX4_ΔC construct as a negative control for C-terminal epitope recognition

• Low signal strength: DUX4 is often expressed at low levels in biological samples. Consider:

  • Extended exposure times

  • Signal amplification using multiple layers (streptavidin-HRP or streptavidin-poly-HRP systems)

  • Enhanced chemiluminescence detection reagents optimized for biotin-streptavidin systems

• Sample preparation considerations: DUX4's unusual charge distribution may affect migration patterns. Use appropriate reducing conditions and SDS concentrations to ensure consistent denaturation.

How can researchers validate experimental findings when studying DUX4-protein interactions?

Proper validation of DUX4 interaction studies requires multiple complementary approaches:

• Control constructs: Use DUX4_ΔC (lacking the C-terminal domain) as a negative control to distinguish interactions specific to the C-terminal activation region versus non-specific or N-terminal-mediated interactions .

• Biological replication: Perform experiments in biological quadruplicates to ensure statistical significance, as demonstrated in published AP-MS analyses of DUX4 .

• Cross-validation methods:

  • Confirm antibody-based findings with aptamer binding assays

  • Use fluorescence techniques (FRET, fluorescence polarization) to quantify binding affinities

  • Apply GST-pulldown assays with purified components to verify direct interactions

• Expected validated interactors: Several well-characterized interactions can serve as positive controls:

  • p300 and CREBBP/CBP histone acetyltransferases

  • Mediator complex components (especially MED15/MED25)

  • H3.X and H3.Y histone variants

How can DUX4 Antibody, Biotin conjugated advance research in muscular dystrophy?

In FSHD research, the DUX4 Antibody, Biotin conjugated offers several methodological advantages:

• Biomarker detection: As aberrant expression of DUX4 leads to FSHD, the biotin-conjugated antibody can serve as a tool for detecting pathological DUX4 expression in muscle biopsy samples.

• Therapeutic development pipeline:

  • Initial screening: Identify compounds that disrupt DUX4's interaction with critical partners like p300/CBP or the Mediator complex

  • Validation: Confirm that disrupting these interactions reduces DUX4 toxicity

  • Mechanism studies: Determine if therapeutic candidates affect DUX4 protein levels, localization, or downstream gene activation

• Combinatorial approaches: Integrate antibody detection with aptamer-based therapeutics that have been shown to specifically target DUX4 with high affinity, potentially revealing novel therapeutic strategies for DUX4-related diseases .

What are emerging applications for studying DUX4's role in developmental biology?

The DUX4 Antibody, Biotin conjugated provides valuable capabilities for developmental biology research:

• Embryonic genome activation studies: DUX4 is markedly enriched in human zygotes with intense nuclear localization preceding minor EGA. The antibody can track this localization pattern in various developmental stages .

• Enhancer activation analysis:

  • DUX4 activates thousands of newly identified transcribed enhancer-like regions

  • These regions are preferentially located within ERVL-MaLR repeat elements

  • The biotin-conjugated antibody can be used to immunoprecipitate and identify these regions in chromatin contexts

• Mediator complex interaction studies: The antibody can be used to investigate DUX4's interaction with the Mediator complex via the C-terminal KIX binding motif, providing insights into the molecular mechanisms of transcriptional activation during early development .

• Chromatin remodeling investigations: DUX4's interaction with histone acetyltransferases elevates levels of acetylated histone H3 in nucleosomes around DUX4 binding sites. The biotin-conjugated antibody can help track these epigenetic modifications in a developmental context .

What methodological approaches can be employed to study DUX4 in oncology research?

For cancer research applications involving DUX4 (implicated in leukemia and sarcomas), researchers should consider:

• Detection in clinical samples: The biotin-conjugated antibody enables sensitive detection of DUX4 in patient samples through multiple techniques (IHC, IF, ICC) .

• Protein interaction profiling in cancer contexts:

  • Perform AP-MS experiments using the biotin-conjugated antibody to identify cancer-specific interaction partners

  • Compare the DUX4 interactome in normal versus malignant cells to identify pathological interactions

  • Investigate how these interactions may contribute to oncogenesis

• Therapeutic targeting validation:

  • Use the antibody to confirm target engagement of potential DUX4-inhibiting compounds

  • Monitor changes in DUX4 localization, activity, or downstream effects following treatment

  • Validate the specificity of novel therapeutics like aptamers designed to neutralize DUX4 function