SOX8 Antibody, Biotin conjugated

What is SOX8 and what cellular processes does it regulate?

SOX8 belongs to the SOX (SRY-related HMG box) family of transcription factors that are related to the mammalian sex determining gene SRY. These genes contain sequences encoding the HMG-box domain responsible for sequence-specific DNA-binding activity. SOX8 functions as a transcriptional regulator implicated in cell fate decisions during development and control of diverse developmental processes. The protein is primarily localized in the nucleus where it exerts its transcriptional activity . SOX8 has been shown to be conserved during evolution and plays key roles in animal development, with some SOX family members involved in human diseases including sex reversal . Recent research has also identified crucial roles for SOX8 in specific cellular contexts, including M cell maturation in intestinal epithelium where it regulates Gp2 expression, which is essential for immunological functions .

What applications is SOX8 Antibody, Biotin conjugated suitable for?

SOX8 Antibody, Biotin conjugated (e.g., catalog #bs-11600R-Biotin) is suitable for multiple experimental applications including:

These applications enable researchers to detect SOX8 protein expression in various sample types and experimental conditions . The biotin conjugation provides enhanced sensitivity through biotin-streptavidin detection systems, which is particularly useful for detecting low abundance proteins or when signal amplification is required. The versatility of this antibody across multiple applications makes it a valuable research tool for studying SOX8 expression and function.

How should SOX8 Antibody, Biotin conjugated be stored and handled to maintain optimal activity?

For optimal preservation of antibody activity, SOX8 Antibody, Biotin conjugated should be stored at -20°C, where it remains stable for up to 12 months . The antibody is supplied in an aqueous buffered solution containing 0.01M TBS (pH 7.4) with 1% BSA, 0.03% Proclin300, and 50% Glycerol . This formulation helps maintain protein stability during storage and prevents microbial contamination.

When handling the antibody, researchers should:

• Avoid repeated freeze-thaw cycles by aliquoting the antibody upon first thaw

• Thaw aliquots at room temperature and briefly centrifuge before use

• Return to -20°C immediately after use

• Work with the antibody on ice when preparing dilutions

• Prepare working dilutions fresh before use rather than storing diluted antibody

These handling practices will help maintain antibody specificity and sensitivity throughout the research project.

How can SOX8 Antibody, Biotin conjugated be utilized in chromatin immunoprecipitation (ChIP) assays to identify SOX8 binding sites?

SOX8 Antibody, Biotin conjugated can be effectively employed in ChIP assays to identify genomic binding sites of SOX8. Research has demonstrated that SOX8 binds to specific promoter regions, such as the GOLPH3 promoter in tongue squamous cell carcinoma (TSCC) cells . When implementing ChIP with this biotin-conjugated antibody, researchers should:

• Crosslink protein-DNA complexes in target cells using formaldehyde (typically 1% for 10 minutes)

• Lyse cells and sonicate chromatin to fragments of 200-500 bp

• Pre-clear chromatin with protein A/G beads

• Capture SOX8-DNA complexes using streptavidin-coated magnetic beads that bind directly to the biotin-conjugated SOX8 antibody

• Wash stringently to remove non-specific binding

• Reverse crosslinks and purify DNA

• Analyze enriched DNA by qPCR, sequencing, or microarray

This methodology has successfully identified that SOX8 binds to the GOLPH3 promoter region within TSCC cell lines, with stronger binding observed in cancerous cell lines (SCC9, HSC3, SCC25, HSC6) compared to normal oral keratinocytes (NOK) . The biotin conjugation provides a significant advantage by eliminating the need for a secondary antibody capture step, potentially reducing background and improving specificity in ChIP experiments.

What approaches can be used to validate SOX8 binding specificity in biotin-streptavidin pull-down assays?

To validate SOX8 binding specificity in biotin-streptavidin pull-down assays, multiple complementary approaches should be employed:

• Control probes validation: Include non-specific probes (NSP) alongside 5'-biotin conjugated target promoter probes. Research on GOLPH3 promoter binding demonstrated minimal SOX8 binding to NSP compared to specific binding to the GOLPH3 promoter .

• Correlation with expression levels: Compare SOX8 binding across cell lines with different SOX8 expression levels. Studies showed stronger SOX8 binding to the GOLPH3 promoter in cell lines with higher SOX8 expression .

• Manipulation of expression levels: Assess binding after SOX8 knockdown or overexpression. Research demonstrated that SOX8 knockdown reduced binding to the GOLPH3 promoter, while overexpression increased binding .

• Luciferase reporter assays: Confirm functional consequences of binding by measuring promoter activity using luciferase reporter constructs containing the putative binding sites. SOX8 overexpression increased GOLPH3 promoter activity, while SOX8 knockdown reduced it .

• Mutational analysis: Create mutations in predicted SOX8 binding sites and assess changes in binding affinity and promoter activity.

This multi-faceted validation approach ensures that detected interactions represent genuine biological activity rather than technical artifacts.

How can SOX8 Antibody, Biotin conjugated be used to investigate SOX8's role in developmental processes?

SOX8 Antibody, Biotin conjugated provides a powerful tool for investigating SOX8's role in developmental processes through several methodological approaches:

• Spatiotemporal expression analysis: Using immunofluorescence with biotin-conjugated SOX8 antibody combined with streptavidin-fluorophore detection, researchers can map SOX8 expression patterns during development. This approach revealed that Sox8 is expressed during all stages of M cell development in the intestinal epithelium .

• Co-localization studies: By combining SOX8 antibody staining with markers of cell differentiation states (like Spi-B and GP2 in M cells), researchers can determine when and where SOX8 is expressed relative to other developmental markers. Studies showed that Sox8 is expressed homogeneously in both immature (Spi-B+GP2-/low) and mature (Spi-B+GP2high) M cells .

• Developmental trajectory mapping: Quantitative image cytometry combined with positional information can reveal how SOX8 expression changes along developmental axes. Research demonstrated that Sox8+ cells were detectable from early stages of M cell differentiation, with expression maintained throughout maturation .

• Genetic models analysis: Comparing wild-type to Sox8-deficient (Sox8-/-) tissues can reveal developmental abnormalities. Studies showed that Sox8-/- mice have significantly fewer GP2+ M cells (0.85±0.09 cells/100μm² compared to 10.4±2.85 cells/100μm² in wild-type), indicating Sox8's essential role in M cell maturation .

• Functional consequences assessment: Beyond structural development, SOX8 antibody can help assess functional outcomes of developmental processes. Research demonstrated that Sox8 deficiency led to attenuated germinal center reactions and antigen-specific IgA responses in mice .

These approaches collectively provide comprehensive insights into SOX8's developmental functions across multiple tissue contexts.

What are the key considerations when using SOX8 Antibody, Biotin conjugated for detecting SOX8 in cancer research?

When employing SOX8 Antibody, Biotin conjugated for cancer research, several key methodological considerations should be addressed:

• Expression heterogeneity: SOX8 expression varies significantly between normal and cancerous tissues. In tongue squamous cell carcinoma (TSCC) research, SOX8 protein was abundant in cancer cell lines (SCC9, HSC3, SCC25, HSC6) but rarely expressed in normal oral keratinocytes (NOK) . This heterogeneity necessitates careful selection of positive and negative controls.

• Signal pathway context: SOX8 functions within complex signaling networks. Research has shown that SOX8 regulates the PI3K/Akt and GSK3β/FOXO1 pathways through GOLPH3 in TSCC . When designing experiments, researchers should consider co-staining for these pathway components to contextualize SOX8 findings.

• Functional validation: Beyond detection, functional validation through knockdown and overexpression experiments is crucial. Studies demonstrated that SOX8 knockdown inhibited TSCC cell proliferation, colony formation, migration, and invasion, while SOX8 overexpression enhanced these processes .

• Biotin-streptavidin system optimization: The biotin-conjugated format requires optimization of streptavidin detection reagents. For immunohistochemistry in tumor tissues, researchers should test multiple streptavidin concentrations and detection systems to minimize background while maximizing specific signal.

• Clinicopathological correlation: Correlate SOX8 expression with patient outcomes and clinicopathological features. Research has shown that high SOX8 expression predicts poor prognosis in TSCC through GOLPH3 signaling .

By addressing these considerations, researchers can maximize the utility of SOX8 Antibody, Biotin conjugated in cancer research contexts.

What are common causes of non-specific binding when using SOX8 Antibody, Biotin conjugated, and how can they be addressed?

Non-specific binding with SOX8 Antibody, Biotin conjugated can arise from several sources, which can be systematically addressed through specific methodological interventions:

• Endogenous biotin interference: Tissues and cells naturally contain biotin, which can lead to false-positive signals when using streptavidin detection systems. To address this:

  • Block endogenous biotin using avidin/biotin blocking kits before antibody application

  • For tissues with high endogenous biotin (e.g., liver, kidney), consider alternative non-biotin detection methods

• Cross-reactivity with related SOX family proteins: The SOX family contains highly homologous members that may cross-react with SOX8 antibodies. To minimize this:

  • Increase antibody dilution to reduce non-specific binding while maintaining specific signal

  • Include appropriate negative controls such as SOX8 knockout/knockdown samples

  • Validate results with a second SOX8 antibody targeting a different epitope

• Non-specific Fc receptor binding: Particularly in immune cells or tissues rich in Fc receptor-expressing cells. To reduce this:

  • Pre-block samples with normal serum from the same species as the secondary reagent

  • Use Fc receptor blocking reagents specific to your sample type

• Suboptimal blocking: Inadequate blocking leads to high background. Address by:

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

  • Using a combination of BSA, normal serum, and non-ionic detergents in blocking buffer

  • Testing alternative blocking reagents like casein or commercial blockers

• Excessive antibody concentration: High antibody concentrations increase non-specific binding. To optimize:

  • Perform titration experiments to determine minimal antibody concentration giving specific signal

  • For SOX8 Antibody, Biotin conjugated, start with the recommended dilutions for each application (e.g., 1:300-5000 for WB) and adjust as needed

By systematically addressing these common causes, researchers can significantly improve the specificity of SOX8 detection using biotin-conjugated antibodies.

How can researchers optimize the biotin-streptavidin detection system when using SOX8 Antibody, Biotin conjugated for different applications?

Optimizing the biotin-streptavidin detection system for SOX8 Antibody, Biotin conjugated requires application-specific strategies:

For Western Blotting:

• Use dilution ranges of 1:300-5000 , starting at the middle range and adjusting based on results

• Employ streptavidin-HRP conjugates with enhanced chemiluminescence (ECL) detection

• For low-abundance samples, consider using streptavidin-poly-HRP systems for signal amplification

• Include 0.05-0.1% Tween-20 in wash buffers to reduce background

• Optimize blocking (5% non-fat milk or 3-5% BSA) based on signal-to-noise ratio

For Immunohistochemistry (IHC-P and IHC-F):

• Use recommended dilutions (1:200-400 for IHC-P; 1:100-500 for IHC-F)

• Test multiple antigen retrieval methods (heat-induced vs. enzymatic)

• Employ streptavidin conjugates with appropriate chromogens or fluorophores

• For fluorescent detection, use streptavidin conjugated to bright, photostable fluorophores

• For chromogenic detection, optimize DAB development time to prevent overstaining

• Consider tyramide signal amplification for detecting low-expression samples

For ELISA:

• Use recommended dilution range of 1:500-1000

• Test multiple plate coating buffers (carbonate/bicarbonate vs. PBS)

• Optimize streptavidin-enzyme (HRP or AP) concentration and development time

• Include 0.05% Tween-20 in wash buffers and 1-3% BSA in diluents

• Consider using streptavidin-poly-HRP for enhanced sensitivity

For Chromatin Immunoprecipitation (ChIP):

• Pre-clear chromatin with streptavidin beads before adding biotin-conjugated antibody

• Use streptavidin magnetic beads for efficient complex capture

• Optimize antibody concentration and incubation time (typically 2-5 μg for 4 hours to overnight)

• Include non-specific biotin-conjugated IgG controls

• Increase wash stringency to reduce background

These application-specific optimizations enhance detection sensitivity and specificity while minimizing background interference.

How can SOX8 Antibody, Biotin conjugated be used to investigate SOX8's role in transcriptional regulation mechanisms?

SOX8 Antibody, Biotin conjugated provides a powerful tool for elucidating SOX8's role in transcriptional regulation through several methodological approaches:

• Chromatin Immunoprecipitation (ChIP): The biotin-conjugated antibody can be used in ChIP assays to identify genomic binding sites of SOX8. Research has demonstrated this approach successfully identified SOX8 binding to the GOLPH3 promoter . The procedure involves:

  • Crosslinking protein-DNA complexes

  • Shearing chromatin to appropriate fragment size

  • Immunoprecipitating with biotin-conjugated SOX8 antibody

  • Capturing complexes with streptavidin beads

  • Analyzing enriched DNA by qPCR or sequencing

• ChIP-seq analysis: Extending the ChIP approach with next-generation sequencing allows genome-wide identification of SOX8 binding sites and revelation of binding motifs. This approach can identify both known and novel SOX8 target genes beyond those already described, such as Gp2 and GOLPH3 .

• Luciferase reporter assays: After identifying potential binding sites, validate functional consequences using reporter constructs with wild-type or mutated SOX8 binding sites. Studies showed SOX8 overexpression increased GOLPH3 promoter activity, while SOX8 knockdown reduced it , confirming direct transcriptional regulation.

• Transcriptome analysis in SOX8-deficient models: Comparative RNA-seq analysis of wild-type versus Sox8-/- samples can identify downstream genes affected by SOX8 deficiency. Research identified 114 significantly down-regulated genes in Sox8-/- FAE compared to controls , providing insight into SOX8's transcriptional network.

• Co-immunoprecipitation with transcriptional co-factors: Using the biotin-conjugated antibody to pull down SOX8 complexes can identify co-factors that collaborate with SOX8 in transcriptional regulation.

Through these approaches, researchers have established that SOX8 directly regulates genes critical for cellular function, such as Gp2 expression in M cell maturation and GOLPH3 in cancer cell proliferation and migration .

What are the recommended controls when using SOX8 Antibody, Biotin conjugated in different experimental settings?

Implementing appropriate controls is critical for validating results with SOX8 Antibody, Biotin conjugated across different experimental platforms:

For Western Blotting:

• Positive control: Lysate from cells/tissues known to express SOX8 (e.g., SCC9, HSC3, SCC25, HSC6 cell lines)

• Negative control: Lysate from cells with minimal SOX8 expression (e.g., normal oral keratinocytes) or Sox8 knockout/knockdown samples

• Loading control: Probe for housekeeping protein (β-actin, GAPDH) to normalize expression

• Molecular weight marker: Confirm detection at expected size (SOX8 molecular weight)

• Primary antibody omission: To detect non-specific binding of detection reagents

For Immunohistochemistry/Immunofluorescence:

• Positive tissue control: Sections known to express SOX8 (e.g., intestinal epithelium containing M cells)

• Negative tissue control: Tissues with no/minimal SOX8 expression or Sox8-/- tissues

• Antibody substitution control: Non-specific IgG from same species (rabbit) at equivalent concentration

• Antigen competition: Pre-incubation of antibody with immunizing peptide to confirm specificity

• Secondary reagent control: Omit primary antibody to detect non-specific binding

For ChIP Assays:

• Input control: Sheared chromatin prior to immunoprecipitation (typically 1-10%)

• Positive locus control: Known SOX8 binding region (e.g., GOLPH3 promoter) or Gp2 promoter

• Negative locus control: Genomic region without SOX8 binding sites

• Isotype control: Biotin-conjugated non-specific IgG immunoprecipitation

• Biological controls: Compare cells with high vs. low SOX8 expression

For Functional Studies:

• Gain-of-function controls: Cells overexpressing SOX8 compared to vector controls

• Loss-of-function controls: SOX8 knockdown/knockout cells compared to scrambled shRNA controls

• Rescue experiments: Reintroduction of SOX8 in knockdown/knockout systems to restore function

• Downstream pathway manipulation: GOLPH3 level restoration assays in SOX8-deficient cells

How should researchers interpret conflicting results between SOX8 expression detected by antibody-based methods versus RNA expression analysis?

When faced with discrepancies between SOX8 protein detection using SOX8 Antibody, Biotin conjugated and RNA expression analysis, researchers should consider several methodological factors and biological mechanisms that may explain these differences:

• Post-transcriptional regulation: SOX8 may be subjected to microRNA-mediated degradation or translational repression, resulting in reduced protein levels despite abundant mRNA. This possibility should be investigated by:

  • Analyzing microRNA binding sites in SOX8 mRNA

  • Assessing SOX8 mRNA association with polysomes versus P-bodies

  • Measuring SOX8 protein half-life in different cellular contexts

• Technical sensitivity differences: Antibody-based protein detection and RNA quantification methods have different detection thresholds. To address this:

  • Compare multiple RNA quantification methods (qRT-PCR, RNA-seq, in situ hybridization)

  • Use multiple antibody detection methods with different sensitivity levels (standard Western blot vs. chemiluminescence vs. fluorescence)

  • Implement more sensitive protein detection methods like proximity ligation assay

• Spatial-temporal discrepancies: The timing of sample collection may capture transcription but miss subsequent translation. This can be addressed by:

  • Performing time-course experiments capturing both RNA and protein at multiple timepoints

  • Using protein synthesis inhibitors to assess protein stability

  • Implementing ribosome profiling to assess translation efficiency

• Cell type heterogeneity: In complex tissues, SOX8 may be expressed in specific cell subpopulations. Research has shown that SOX8 is specifically expressed in M cells within intestinal epithelium , which might be diluted in whole-tissue RNA preparations. To address this:

  • Perform single-cell RNA sequencing to identify cell-specific expression patterns

  • Use laser capture microdissection to isolate specific cell populations before analysis

  • Implement co-localization studies with cell-type-specific markers

• Antibody cross-reactivity: The SOX8 antibody may cross-react with related SOX family proteins. This possibility should be evaluated by:

  • Testing the antibody in Sox8-/- samples as negative controls

  • Performing peptide competition assays

  • Comparing results with alternative SOX8 antibodies targeting different epitopes

By systematically addressing these possibilities, researchers can resolve apparent discrepancies and gain deeper insights into SOX8 regulation and function.

What statistical approaches are recommended for quantifying SOX8 expression in immunohistochemistry applications using SOX8 Antibody, Biotin conjugated?

To rigorously quantify SOX8 expression in immunohistochemistry using SOX8 Antibody, Biotin conjugated, several statistical approaches should be implemented:

• Whole-mount quantitative image cytometry: This approach has been successfully used to analyze Sox8 expression in follicle-associated epithelium . The methodology involves:

  • Acquiring high-resolution images with consistent exposure settings

  • Defining regions of interest objectively (e.g., entire FAE dome)

  • Using automated cell detection with appropriate size and intensity thresholds

  • Measuring fluorescence intensity in individual cells

  • Implementing quartile-based intensity categorization (negative, low, medium, high)

• Spatial distribution analysis: Because SOX8 expression may vary along developmental or anatomical gradients, spatial statistical methods can provide valuable insights:

  • Measure distances from anatomical landmarks (e.g., from crypt bottom to SOX8+ cells)

  • Plot expression intensity against spatial coordinates

  • Implement heat maps to visualize expression patterns

  • Apply spatial autocorrelation statistics (Moran's I, Geary's C) to quantify clustering

• Co-expression correlation analysis: For multiparameter staining with other markers:

  • Calculate Pearson's or Spearman's correlation coefficients between SOX8 and other markers

  • Apply co-localization coefficients (Manders' overlap coefficient)

  • Implement nearest neighbor analysis for spatial relationships between different cell types

  • Use conditional probability analysis to determine marker co-occurrence frequencies

• Cell counting approaches:

  • Express SOX8+ cells per unit area (e.g., cells/100 μm²) as implemented in Sox8-/- studies

  • Use stereological principles for unbiased counting (optical disectors)

  • Implement automated cell counting algorithms with appropriate validation

  • Always include inter-observer reliability assessments

• Statistical testing and power analysis:

  • Use appropriate statistical tests based on data distribution (parametric vs. non-parametric)

  • Implement mixed-effects models to account for within-subject correlations

  • Conduct power analysis to determine adequate sample size (typically n≥5 samples per group)

  • Report effect sizes (Cohen's d, Hedge's g) in addition to p-values

  • Use multiple comparison corrections for analyses involving numerous markers or regions

These rigorous quantitative approaches enable objective assessment of SOX8 expression patterns and their relationships to biological processes and disease states.

What are emerging applications for SOX8 Antibody, Biotin conjugated in cutting-edge research techniques?

SOX8 Antibody, Biotin conjugated is poised to play important roles in several emerging research techniques that promise to deepen our understanding of SOX8 biology:

• Spatial transcriptomics integration: Combining antibody-based SOX8 protein detection with spatial transcriptomics can reveal relationships between SOX8 protein localization and broader transcriptional landscapes. This approach would be particularly valuable for understanding SOX8's role in heterogeneous tissues like intestinal epithelium, where SOX8 is specifically expressed in M cells .

• Proximity-dependent biotinylation (BioID/TurboID): The biotin conjugation of this antibody can be leveraged in two-step approaches where SOX8 is first immunoprecipitated and then used to identify proximal proteins. Alternatively, researchers could develop SOX8-BioID fusion proteins to map the SOX8 interactome in living cells, providing insights into its transcriptional complexes and regulatory partners.

• CUT&Tag and CUT&RUN applications: These emerging alternatives to traditional ChIP-seq offer higher sensitivity and lower background. The biotin-conjugated SOX8 antibody could be adapted to these protocols to map SOX8 genomic binding sites with improved resolution, potentially revealing subtle binding patterns missed by conventional ChIP-seq.

• Live-cell imaging with fluorescent streptavidin: By using cell-permeable biotin-conjugated antibody fragments and fluorescent streptavidin, researchers could potentially track SOX8 dynamics in living cells, providing insights into its nuclear translocation, retention, and turnover in response to developmental or oncogenic signals.

• Antibody-guided CRISPR perturbations: Emerging techniques like CRISPR-HOT could utilize the biotin-conjugated SOX8 antibody to guide Cas9 to SOX8-bound genomic regions, enabling targeted perturbation of SOX8 activity at specific loci rather than global SOX8 knockout.

These emerging applications have the potential to address fundamental questions about SOX8 function, including how it selects genomic targets, how its activity is regulated post-translationally, and how it interacts with other factors to control gene expression in normal development and disease states.

Based on current research, what are the most promising directions for future studies utilizing SOX8 Antibody, Biotin conjugated?

Based on the reviewed literature, several high-priority research directions emerge for future studies utilizing SOX8 Antibody, Biotin conjugated:

• Cancer biomarker development: Given SOX8's role in promoting tumor growth and poor prognosis through GOLPH3 signaling in tongue squamous cell carcinoma , further investigation of SOX8 as a diagnostic or prognostic biomarker is warranted. The biotin-conjugated antibody could be employed in:

  • Tissue microarray studies across multiple cancer types

  • Circulating tumor cell detection methods

  • Multiplexed imaging with other oncogenic markers

• Immune system development and modulation: The essential role of SOX8 in M cell maturation and subsequent IgA responses suggests broader implications for mucosal immunity. Future studies should explore:

  • SOX8 expression in other mucosal immune cell populations

  • The impact of SOX8 modulation on mucosal vaccine efficacy

  • SOX8's role in pathogen uptake and immune response initiation

• Developmental biology applications: The conserved role of SOX8 in development suggests unexplored functions in cell fate determination. Research could focus on:

  • SOX8 expression dynamics during embryonic development

  • SOX8's role in stem cell maintenance and differentiation

  • Comparative analysis of SOX8 function across species

• Therapeutic target validation: Based on findings that SOX8 knockdown inhibits cancer cell proliferation, invasion, and migration , SOX8 represents a potential therapeutic target. Studies could explore:

  • Development of small molecule inhibitors of SOX8-DNA interaction

  • Analysis of SOX8-dependent transcriptional networks as therapeutic targets

  • Evaluation of SOX8 regulation as a means to modulate cancer progression

• Multi-omics integration: Combining SOX8 protein detection with transcriptomics, proteomics, and epigenomics could reveal comprehensive insights into SOX8 function:

  • Integrated ChIP-seq, ATAC-seq, and RNA-seq in SOX8-manipulated systems

  • Correlation of SOX8 binding with histone modifications and chromatin accessibility

  • Identification of SOX8-dependent epigenetic changes during cell differentiation

These research directions would significantly expand our understanding of SOX8 biology while leveraging the specificity and sensitivity of biotin-conjugated SOX8 antibody for various detection and functional studies.