SOX4 Antibody

What is SOX4 and why is it a significant target for research antibodies?

SOX4 is a transcription factor belonging to the SOX (SRY-related HMG-box) family that plays critical roles in embryonic development, cell-fate determination, and organogenesis of multiple tissues including heart, pancreas, brain, and lymphocyte differentiation. Its significance as a research target stems from its involvement in multiple cellular processes:

• Regulates transcription by binding to the T-cell enhancer motif 5'-AACAAAG-3'

• Functions as a downstream target of TGF-β signaling

• Shows upregulated expression in numerous cancer types, contributing to cellular transformation, survival, and metastasis

• Acts as a master regulator of epithelial-mesenchymal transition (EMT)

• Plays roles in immune cell development and differentiation

Antibodies targeting SOX4 are therefore essential tools for investigating these diverse biological processes and their dysregulation in disease states.

How do I select the appropriate SOX4 antibody for my specific research application?

Selection of an appropriate SOX4 antibody requires consideration of several experimental parameters:

• Species reactivity: Confirm cross-reactivity with your species of interest. Available antibodies show reactivity to human, mouse, and rat SOX4 .

• Application compatibility: Verify suitability for your intended application. Available SOX4 antibodies have been validated for:

  • Immunohistochemistry-paraffin (IHC-P)

  • Immunocytochemistry/Immunofluorescence (ICC/IF)

  • Western blotting (availability varies by vendor)

• Clonality consideration:

  • Monoclonal antibodies (e.g., clone CL5634, SOX4/2540) offer high specificity and reproducibility for most applications

  • Consider the immunogen used to generate the antibody (synthetic peptides vs. recombinant full-length protein)

• Subcellular localization: Since SOX4 is predominantly nuclear, select antibodies validated for nuclear detection in immunostaining applications

• Special considerations: When using mouse antibodies on mouse tissues, employ Mouse-on-Mouse blocking reagents to reduce background signal

What are the key differences between SOX4 and other SOX family members that affect antibody specificity?

Understanding SOX4's structural uniqueness is critical for antibody selection:

• Domain structure distinctions:

  • SOX4 belongs to the group C subfamily (with SOX11 and SOX12)

  • Contains a highly conserved HMG-box DNA-binding domain shared with other SOX proteins

  • Features unique N-terminal and C-terminal regions that distinguish it from other SOX family members

  • The HMG box and C-terminal portion are critical for interactions with other proteins like GATA-3

• Antibody cross-reactivity concerns:

  • Antibodies targeting the HMG-box may cross-react with other SOX family members

  • Antibodies generated against N- or C-terminal regions offer higher specificity

  • Validation for specificity is crucial, particularly in tissues that express multiple SOX family members (e.g., developing nervous system expresses both SOX4 and SOX11)

• Functional region targeting:

  • Mutations in specific regions (e.g., Arg61/Pro62 or Phe66/Met67) affect SOX4's ability to interact with partner proteins like GATA-3

  • Consider antibodies that don't mask functional domains when studying protein-protein interactions

How can SOX4 antibodies be used to study its role in tumor progression and metastasis?

SOX4 antibodies provide valuable tools for investigating various aspects of tumor biology:

• Expression level analysis:

  • IHC-P analysis of patient samples shows SOX4 upregulation in 24 different tumor types, particularly in liver hepatocellular carcinoma (LIHC)

  • Western blot quantification can correlate SOX4 expression levels with clinical outcomes and treatment responses

• Cellular localization studies:

  • ICC/IF techniques reveal subcellular distribution of SOX4 in cancer cells

  • Nuclear accumulation can be quantified and correlated with transcriptional activity

• Tumor microenvironment interactions:

  • IHC multiplex staining can examine SOX4 expression in relation to immune infiltration patterns

  • SOX4 expression correlates with tumor immune microenvironment (TIME) characteristics in LIHC

• Metastasis research:

  • SOX4 antibodies can track expression in primary tumors versus metastatic sites

  • SOX4 is included in gene signatures associated with breast cancer metastasis to lung and brain

• Methodological protocol:

  • For patient-derived xenografts: Fix tissues in 10% formalin, embed in paraffin, section at 4-5μm

  • Perform heat-mediated antigen retrieval (pH 6.0 citrate buffer recommended)

  • Block with 5% normal serum, incubate with primary SOX4 antibody (2μg/ml for ab236557)

  • Visualize using appropriate detection system; counterstain nuclei with hematoxylin

What is the significance of SOX4 in TGF-β-mediated epithelial-to-mesenchymal transition (EMT) and how can antibodies help investigate this process?

SOX4 plays a crucial role in TGF-β-induced EMT, representing a significant area for antibody-based investigation:

• SOX4-SMAD3 interaction analysis:

  • Co-immunoprecipitation (Co-IP) using SOX4 antibodies can detect SOX4-SMAD3 complexes

  • SOX4 has been identified as a novel, functionally relevant SMAD3 interaction partner

  • This interaction is cell-type specific and affects transcriptional outcomes

• Chromatin immunoprecipitation (ChIP) applications:

  • SOX4 antibodies enable ChIP assays to identify genomic loci co-occupied by SOX4 and SMAD3

  • ChIP-seq analysis reveals that SOX4 and SMAD3 co-occupy a large number of genomic loci in a cell-type specific manner

  • Methodologically, this requires cross-linking cells with 1% formaldehyde, sonication to fragment chromatin, and immunoprecipitation with SOX4 antibodies

• EMT marker correlation studies:

  • Multiplexed IF using SOX4 antibodies alongside EMT markers (E-cadherin, vimentin, etc.)

  • SOX4 expression is indispensable for EMT and cell survival in vitro and for primary tumor growth and metastasis in vivo

• Lenvatinib resistance mechanism:

  • SOX4 antibodies can monitor expression changes in response to treatments

  • Lenvatinib treatment increases SOX4 expression in hepatocellular carcinoma cells, contributing to drug resistance

  • Silencing SOX4 effectively eliminates drug resistance caused by lenvatinib treatment

• EZH2 regulation pathway:

  • SOX4 governs expression of the epigenetic modifier EZH2, which mediates EMT

  • Dual immunostaining for SOX4 and EZH2 can reveal this regulatory axis

How can SOX4 antibodies be incorporated into prognostic models for cancer?

SOX4 expression analysis using antibodies provides valuable prognostic information:

• Tissue microarray (TMA) applications:

  • SOX4 antibody staining of TMAs allows high-throughput analysis of expression across multiple patient samples

  • Scoring systems can be developed based on staining intensity and percentage of positive cells

  • SOX4 expression is strongly associated with unfavorable prognoses across different tumor types

• Integration with DNA methylation data:

  • Combine SOX4 IHC with methylation analysis of SOX4 promoter

  • SOX4 expression correlates with DNA methylation levels across different tumor types

  • Specific methylation sites correlate with SOX4 expression in LIHC

• Correlation with tumor mutation burden (TMB):

  • SOX4 expression has been analyzed in relation to TMB and microsatellite instability (MSI) across TCGA tumors

  • Understanding these relationships can inform immunotherapy response prediction

• Multi-parameter prognostic models:

  • Six favorable prognostic models incorporate SOX4-associated genes to predict LIHC prognosis

  • These models integrate multiple parameters including expression of DNA damage repair, EMT, m6A methylation, hypoxia, ferroptosis, and energy-metabolism-related genes

• Methodological approach:

  • Standardize IHC protocols using automated staining platforms

  • Apply digital pathology and image analysis for quantitative assessment

  • Use statistical models (Cox regression, nomograms) to integrate SOX4 expression with clinical parameters

What is the role of SOX4 in T helper cell differentiation and how can antibodies help study this process?

SOX4 functions as a critical regulator of T helper (TH) cell differentiation that can be studied with antibodies:

• TGF-β-mediated SOX4 induction:

  • TGF-β treatment induces significant upregulation of SOX4 mRNA and protein in CD4+ T cells

  • Western blot analysis with SOX4 antibodies can quantify this induction

  • The expression of SOX4 is highest in induced regulatory T (iTreg) cells and lowest in TH2 cells

• SOX4-GATA-3 interaction:

  • Co-immunoprecipitation with SOX4 antibodies enables detection of SOX4-GATA-3 physical association

  • Sox4 inhibits GATA-3 function through direct binding, preventing GATA-3 binding to consensus DNA sequences

  • Both the HMG box and C-terminal portion of SOX4 are important for this association

• Chromatin binding dynamics:

  • ChIP assays using SOX4 antibodies reveal that SOX4 binds to the promoter region of IL5

  • SOX4 prevents binding of GATA-3 to this promoter, thereby inhibiting TH2 differentiation

  • TGF-β treatment induces binding of SOX4 to the IL5 promoter but not to other GATA-3 target sites

• Experimental methodology:

  • Isolate naive CD4+ T cells using magnetic separation

  • Culture under TH2-polarizing conditions with/without TGF-β

  • Fix cells for ChIP or prepare lysates for Western blot/IP using SOX4 antibodies

  • For functional studies, perform cytokine ELISAs or intracellular cytokine staining

How can SOX4 antibodies be used to investigate ectopic lymphoid-like structures (ELSs) formation in inflammatory diseases?

SOX4 antibodies offer valuable tools for studying ELS formation in inflammatory conditions:

• SOX4 expression in synovial CD4+ T cells:

  • IHC or IF staining of synovial tissues from rheumatoid arthritis (RA) patients reveals SOX4 upregulation

  • SOX4 is significantly upregulated in synovial CD4+ T cells compared to blood CD4+ T cells from RA patients

  • Expression correlates with ELS formation in RA synovium

• PD-1hiCXCR5-CD4+ T cell identification:

  • Multiplex immunofluorescence combining SOX4 with PD-1 and CXCR5 antibodies

  • This identifies SOX4+ cells that contribute to ELS formation through CXCL13 production

  • These cells are distinct from follicular helper T (Tfh) cells in secondary lymphoid organs

• CXCL13 co-expression analysis:

  • Double immunostaining for SOX4 and CXCL13 demonstrates their relationship

  • Sox4 functions as a key transcription factor for CXCL13 production in human CD4+ T cells under inflammatory conditions

• TGF-β response element mapping:

  • ChIP-seq using SOX4 antibodies can map binding sites in relation to CXCL13 regulatory regions

  • TGF-β signaling induces Sox4, which contributes to CXCL13 transcription

• Methodological considerations:

  • For synovial tissue analysis: Obtain fresh synovial biopsies, fix in formalin, and process for IHC

  • Optimize antigen retrieval methods (preferably heat-induced epitope retrieval)

  • Consider dual staining methods to correlate SOX4 with functional markers

  • Quantify SOX4+ cells in relation to ELS formation using digital image analysis

What are the optimal protocols for performing ChIP-seq with SOX4 antibodies?

ChIP-seq with SOX4 antibodies requires careful optimization:

• Cell preparation and crosslinking:

  • Harvest 10-20 million cells per ChIP reaction

  • Crosslink with 1% formaldehyde for 10 minutes at room temperature

  • Quench with 0.125M glycine for 5 minutes

  • Wash cells with cold PBS containing protease inhibitors

• Chromatin fragmentation optimization:

  • Lyse cells in appropriate buffers (containing SDS, EDTA, and protease inhibitors)

  • Sonicate to generate 200-500bp DNA fragments

  • Verify fragmentation efficiency by agarose gel electrophoresis

  • Optimize sonication conditions for each cell type

• Immunoprecipitation considerations:

  • Pre-clear chromatin with protein A/G beads

  • Use 2-5μg of SOX4 antibody per reaction

  • Include appropriate isotype controls

  • Incubate overnight at 4°C with rotation

• Washing and elution protocols:

  • Perform stringent washes to remove non-specific binding

  • Elute DNA-protein complexes with elution buffer containing SDS

  • Reverse crosslinking by incubation at 65°C overnight

  • Treat with RNase A and Proteinase K

• Library preparation and sequencing:

  • Purify DNA using column-based methods

  • Prepare libraries according to sequencing platform requirements

  • Include input controls for normalization

  • Use appropriate peak calling algorithms (MACS2 recommended)

• Data validation approaches:

  • Validate peaks by ChIP-qPCR

  • Compare binding sites with published datasets

  • Correlate with transcriptomic data

  • Perform motif analysis to identify SOX4 binding motifs

How can SOX4 antibodies be used in single-cell analysis techniques?

SOX4 antibodies can be adapted for cutting-edge single-cell applications:

• Single-cell immunostaining optimization:

  • For adherent cells: Fix with 4% paraformaldehyde, permeabilize with 0.1% Triton X-100

  • For suspension cells: Fix with 2% paraformaldehyde, permeabilize with methanol or saponin

  • Use fluorophore-conjugated SOX4 antibodies (CF®488A, CF®568 conjugates available)

  • Include nuclear counterstain (DAPI or Hoechst)

• Mass cytometry (CyTOF) considerations:

  • Conjugate SOX4 antibodies with rare earth metals

  • Include in panels with surface markers and other transcription factors

  • Optimize fixation and permeabilization for nuclear factor detection

  • Develop appropriate gating strategies for SOX4+ cell populations

• Single-cell Western blotting:

  • Adapt protocols for microfluidic-based single-cell Western systems

  • Optimize lysis conditions to retain nuclear proteins

  • Determine appropriate antibody dilutions for limited protein amounts

  • Include housekeeping controls for normalization

• Imaging mass cytometry:

  • Use metal-conjugated SOX4 antibodies for spatial analysis in tissues

  • Combine with lineage markers and functional readouts

  • Optimize signal-to-noise ratio for nuclear detection

  • Apply unsupervised clustering algorithms for cell phenotype identification

• Proximity ligation assay (PLA):

  • Use SOX4 antibodies with antibodies against interaction partners (e.g., SMAD3, GATA-3)

  • Visualize protein-protein interactions at single-molecule resolution

  • Quantify interaction frequencies in different cellular contexts

  • Apply in tissue sections to map interaction landscapes

What are the considerations for co-immunoprecipitation experiments involving SOX4?

Successful co-immunoprecipitation of SOX4 and its binding partners requires careful optimization:

• Lysis buffer composition:

  • Use gentle non-ionic detergents (0.5-1% NP-40 or 0.5% Triton X-100)

  • Include protease and phosphatase inhibitors

  • Optimize salt concentration (150-300mM NaCl) to preserve interactions

  • Consider nuclear extraction buffers for efficient SOX4 extraction

• Antibody selection criteria:

  • Choose antibodies that don't interfere with protein-protein interaction domains

  • The HMG box and C-terminal portion of SOX4 are important for protein interactions

  • Consider epitope accessibility in native protein complexes

  • Use antibodies validated for immunoprecipitation applications

• Experimental controls:

  • Include isotype control antibodies

  • Perform reverse immunoprecipitation where possible

  • Use lysates from cells with SOX4 knockdown as negative controls

  • Consider competition with immunizing peptide

• Detection strategies:

  • Western blot using antibodies against predicted interaction partners

  • Mass spectrometry for unbiased identification of binding partners

  • Sequential immunoblotting to detect multiple proteins

  • Stripping and reprobing membranes for technical replicates

• Application to specific interactions:

  • SOX4-SMAD3: Use nuclear extracts from TGF-β-treated cells

  • SOX4-GATA-3: Include both zinc-finger domains of GATA-3

  • Novel interactions: Consider crosslinking to stabilize transient interactions

How can non-specific binding be minimized when using SOX4 antibodies in immunohistochemistry?

Addressing non-specific binding requires methodical optimization:

• Species-specific considerations:

  • When using mouse anti-SOX4 antibodies on mouse tissues, employ Mouse-on-Mouse blocking reagents

  • Mouse-On-Mouse blocking reagent may be needed for IHC and ICC experiments to reduce high background signal

  • Commercial reagents like PK-2200-NB and MP-2400-NB are available for this purpose

• Blocking protocol optimization:

  • Extend blocking time to 1-2 hours at room temperature

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

  • Add 0.1-0.3% Triton X-100 to blocking buffer for better penetration

  • Consider adding 1% BSA to reduce hydrophobic interactions

• Antibody dilution optimization:

  • Perform titration experiments to determine optimal concentration

  • Start with manufacturer's recommended dilution (e.g., 2μg/ml for ab236557)

  • Prepare dilutions in blocking buffer rather than plain buffer

  • Consider overnight incubation at 4°C instead of shorter times at room temperature

• Antigen retrieval method selection:

  • Compare heat-induced epitope retrieval methods (citrate pH 6.0 vs. EDTA pH 9.0)

  • Optimize retrieval time and temperature

  • Consider enzymatic retrieval for certain tissue types

  • Allow slides to cool slowly after heat retrieval

• Secondary antibody considerations:

  • Use highly cross-adsorbed secondary antibodies

  • Consider fluorophore-conjugated secondary antibodies for better signal-to-noise ratio

  • Include secondary-only controls to assess non-specific binding

  • Use detection systems with amplification capabilities for weak signals

What are the critical factors for successful detection of SOX4 in Western blotting?

Western blot optimization for SOX4 detection includes several critical parameters:

• Sample preparation optimization:

  • Use specialized nuclear extraction protocols

  • Include protease inhibitors to prevent degradation

  • Maintain cold temperatures throughout processing

  • Sonicate briefly to shear genomic DNA and release nuclear proteins

• Gel percentage and transfer considerations:

  • Use 10-12% polyacrylamide gels for SOX4 (~47-66 kDa depending on species)

  • Optimize transfer conditions for nuclear proteins (longer transfer times)

  • Consider semi-dry transfer for more efficient transfer of transcription factors

  • Verify transfer efficiency with reversible protein stains

• Blocking and antibody incubation:

  • Block with 5% non-fat dry milk or 3-5% BSA in TBST

  • Dilute primary antibody in blocking buffer

  • Incubate with primary antibody overnight at 4°C

  • Wash extensively (4-5 times) with TBST before secondary antibody

• Signal detection optimization:

  • Use enhanced chemiluminescence (ECL) detection for most applications

  • Consider fluorescent secondary antibodies for multiplexing and quantification

  • Optimize exposure times to avoid saturation

  • Include positive controls (cell lines with known SOX4 expression)

• Troubleshooting common issues:

  • Multiple bands: Verify specificity with blocking peptides

  • No signal: Check protein extraction efficiency from nuclear fraction

  • High background: Increase washing steps and dilute antibody further

  • Inconsistent results: Standardize lysate preparation and protein loading

How should SOX4 antibodies be validated for experimental reproducibility and specificity?

Comprehensive validation ensures reliable experimental outcomes:

• Genetic validation approaches:

  • Test antibodies on SOX4 knockout/knockdown samples

  • Compare with overexpression systems (transient transfection)

  • Use siRNA or shRNA against SOX4 as negative controls

  • Verify correlation between mRNA and protein levels

• Cross-reactivity assessment:

  • Test on tissues known to express multiple SOX family members

  • Perform peptide competition assays

  • Compare staining patterns with orthogonal detection methods

  • Test across multiple species to confirm epitope conservation

• Application-specific validation:

  • For IHC: Compare staining patterns across multiple tissue types

  • For Western blot: Verify molecular weight and band pattern

  • For ChIP: Validate enrichment at known SOX4 target genes

  • For IF: Confirm nuclear localization and compare with RNA expression

• Lot-to-lot consistency testing:

  • Maintain reference samples for comparison

  • Document detailed protocols for each application

  • Consider developing standardized positive controls

  • Archive images/data from validated lots

• Independent validation methods:

  • Compare results using antibodies targeting different epitopes

  • Correlate with SOX4 mRNA expression data

  • Use tagged SOX4 constructs as parallel controls

  • Consider mass spectrometry validation for key findings