SOX30 Antibody

What is SOX30 and what are its primary cellular functions?

SOX30 (Sex-determining region Y-box containing gene 30) is a transcription factor that can function as both a transcriptional activator and repressor. It binds to the DNA sequence 5'-ACAAT-3' with a preference for guanine residues surrounding this core motif . SOX30 plays multiple roles in cellular function, including:

• Binding to its own promoter to activate its own transcription

• Activating expression of postmeiotic genes involved in spermiogenesis

• Binding to the promoter region of CTNNB1 and repressing its transcription, which leads to inhibition of Wnt signaling

• Inhibiting Wnt signaling by binding to the CTNNB1 protein, preventing interaction of CTNNB1 with TCF7L2/TCF4

• Directly binding to the CACTTTG sequence (+115 to +121) of the p53 promoter region and activating p53 transcription

These molecular mechanisms contribute to SOX30's role in regulating cell proliferation, apoptosis, and tumor suppression.

What is the expression pattern of SOX30 in normal versus cancerous tissues?

SOX30 shows differential expression patterns between normal and cancerous tissues. In normal conditions, SOX30 is expressed in tissues such as testis, while its expression is significantly altered in various cancer types:

• In colorectal cancer (CRC), both SOX30 protein and mRNA expressions are reduced in tumor tissue compared to adjacent normal tissue (mean IHC score: 2.6 ± 1.6 vs. 5.5 ± 2.6, p<0.001)

• In lung cancer, SOX30 hypermethylation was detected in 100% of lung cancer cell lines (9/9) and 70.83% (85/120) of primary lung tumor tissues compared with none (0/20) of normal tissues

• Similar downregulation patterns have been observed in breast cancer, non-small cell lung cancer, and hepatocellular carcinoma

The reduced expression in cancer tissues is likely related to SOX30's tumor-suppressive function, with epigenetic silencing through hypermethylation being a primary mechanism for SOX30 inactivation in cancer.

What are the key considerations when selecting a SOX30 antibody for research?

When selecting a SOX30 antibody, researchers should consider several important factors:

• Antibody type and specificity: Current commercial options include rabbit polyclonal antibodies that recognize human SOX30 . Verify the immunogen sequence to ensure it targets the region of interest within SOX30 (common immunogens include recombinant fragments of human SOX30, such as aa 450-600 or specific peptide sequences ).

• Validated applications: Confirm the antibody has been validated for your intended application (IHC-P, WB) .

• Species reactivity: Most available antibodies are validated for human samples; cross-reactivity with other species should be experimentally verified .

• Format and conjugation: Consider whether unconjugated or conjugated formats are needed for your application.

• Supporting validation data: Review the manufacturer's data showing specificity, such as Western blot analysis with positive and negative controls (e.g., vector-only vs. SOX30 overexpression lysates) .

Proper antibody selection is critical for ensuring reliable and reproducible results in SOX30 research.

What are the optimal protocols for using SOX30 antibodies in immunohistochemistry?

For optimal immunohistochemical detection of SOX30 in formalin-fixed paraffin-embedded (FFPE) tissues, the following protocol is recommended:

• Antigen retrieval: HIER (Heat-Induced Epitope Retrieval) at pH 6 is recommended for SOX30 detection .

• Blocking: Use appropriate blocking solution to reduce non-specific binding.

• Primary antibody incubation: Dilute SOX30 antibody to 1:50-1:200 . For ab272553, a 1:50 dilution has been validated , while NBP1-86503 can be used between 1:50-1:200 . Incubate according to manufacturer's recommendations.

• Secondary antibody: Use an appropriate species-specific secondary antibody (e.g., goat anti-rabbit IgG for rabbit primary antibodies) .

• Detection system: Apply a suitable chromogenic or fluorescent detection system.

• Controls: Include positive controls (testis tissue has shown positive staining ) and negative controls (liver and skin tissues have shown negative staining ).

This protocol has been successfully used to evaluate SOX30 expression in colorectal cancer tissues and other tumor types, allowing for semi-quantitative scoring based on staining intensity and density .

What is the recommended approach for Western blot analysis of SOX30?

For effective Western blot detection of SOX30, the following methodological approach is recommended:

• Sample preparation: Prepare protein lysates from tissues or cell lines of interest. SOX30-overexpressing HEK293T cells can serve as a positive control, while vector-only transfected cells can serve as a negative control .

• Protein loading and separation: Load 20-30 μg of protein per lane and separate by SDS-PAGE.

• Transfer and blocking: Transfer proteins to a PVDF or nitrocellulose membrane and block with appropriate blocking buffer.

• Antibody dilution: Use SOX30 antibody at a concentration of 0.4 μg/ml for Western blot applications .

• Detection: Apply appropriate secondary antibody and develop using chemiluminescence or other detection methods.

• Result interpretation: The predicted molecular weight of SOX30 is approximately 82 kDa . Verify band specificity by comparing with controls.

• Validation approach: For additional verification, correlation between Western blot and IHC results can be performed, as demonstrated in CRC studies where SOX30 detected by Western blot showed high correlation with SOX30 detected by IHC .

This approach enables reliable detection and quantification of SOX30 protein expression in experimental samples.

How can SOX30 antibodies be utilized to investigate tumor progression mechanisms?

SOX30 antibodies serve as valuable tools for investigating tumor progression mechanisms through multiple experimental approaches:

• Expression correlation studies: Use SOX30 antibodies in IHC to evaluate expression levels across tumor stages. Studies have demonstrated that SOX30 expression negatively correlates with:

  • Lymph node metastasis

  • T stage

  • N stage

  • TNM stage in colorectal cancer patients

• Pathway analysis: Examine the relationship between SOX30 and Wnt signaling components by co-immunostaining or co-immunoprecipitation, as SOX30 has been shown to repress tumor metastasis by regulating the Wnt signaling pathway .

• Epithelial-mesenchymal transition (EMT) studies: Investigate SOX30's role in modulating EMT processes in cancer progression, particularly in ovarian cancer where SOX30 has been shown to play an anti-metastatic role through EMT regulation .

• p53 regulation mechanism: Use SOX30 antibodies in chromatin immunoprecipitation (ChIP) assays to confirm SOX30 binding to the p53 promoter region, as SOX30 directly binds to CACTTTG (+115 to +121) of the p53 promoter and activates p53 transcription .

• Functional validation: Combine antibody-based detection with gain/loss-of-function studies. For example, researchers have shown that ectopic expression of SOX30 induces cancer cell apoptosis and inhibits proliferation, while knockdown demonstrates reversed effects .

These approaches allow researchers to elucidate SOX30's mechanistic role in tumor suppression and progression.

What is the significance of SOX30 as a prognostic biomarker in cancer research?

SOX30 has emerged as a potential prognostic biomarker in multiple cancer types, with significant research implications:

When investigating SOX30 as a prognostic biomarker, researchers should employ standardized IHC protocols with validated antibodies and scoring systems, such as the semi-quantitative scoring method combining intensity (0-3) and density (0-4) scores used in CRC studies .

How can researchers effectively study SOX30 epigenetic regulation in cancer?

To effectively study SOX30 epigenetic regulation in cancer, researchers can employ the following methodological approaches:

• DNA methylation analysis:

  • Utilize bisulfite sequencing to examine methylation status of the SOX30 promoter region

  • Employ methylation-specific PCR (MSP) to detect SOX30 hypermethylation in tumor samples

  • Consider genome-wide methylation screening approaches that identified SOX30 as a preferentially methylated gene in lung cancer (detected in 100% of lung cancer cell lines and 70.83% of primary tumors)

• Demethylation experiments:

  • Treat cancer cell lines exhibiting SOX30 silencing with demethylating agents (e.g., 5-aza-2'-deoxycytidine)

  • Confirm restoration of SOX30 expression following demethylation using both mRNA and protein analysis (RT-PCR and Western blot/IHC with SOX30 antibodies)

  • This approach confirmed that SOX30 silencing is regulated by hypermethylation in lung cancer

• Correlation studies:

  • Analyze the relationship between SOX30 methylation status and expression levels in patient samples

  • Compare methylation patterns between tumor and adjacent normal tissues

• Functional consequences:

  • Examine how epigenetic silencing of SOX30 impacts downstream pathways (p53 activation, Wnt signaling)

  • Investigate whether SOX30 methylation status correlates with clinical outcomes

• Therapeutic implications:

  • Test whether epigenetic modifying drugs can restore SOX30 expression and tumor-suppressive functions

  • Evaluate potential combinatorial approaches with conventional therapies

This comprehensive approach can reveal the mechanisms and significance of SOX30 epigenetic silencing in cancer development and progression.

What are common challenges and solutions when using SOX30 antibodies for immunohistochemistry?

Researchers may encounter several challenges when using SOX30 antibodies for immunohistochemistry, along with these recommended solutions:

• Weak or absent staining:

  • Problem: Inadequate antigen retrieval or fixation issues

  • Solution: Optimize heat-induced epitope retrieval (HIER) at pH 6 as specifically recommended for SOX30 ; extend retrieval time if necessary

  • Problem: Insufficient antibody concentration

  • Solution: Titrate antibody concentration (test range from 1:50 to 1:200); for challenging samples, use the higher concentration (1:50) as validated in published studies

• High background or non-specific staining:

  • Problem: Insufficient blocking or cross-reactivity

  • Solution: Extend blocking time; use species-specific blocking reagents; include additional washing steps

  • Problem: Antibody concentration too high

  • Solution: Perform antibody titration experiments to determine optimal concentration

• Inconsistent staining across samples:

  • Problem: Variable fixation or processing

  • Solution: Standardize tissue collection and fixation protocols; consider tissue microarrays for comparative studies

  • Problem: Heterogeneous SOX30 expression

  • Solution: Analyze multiple fields and use semi-quantitative scoring methods as described for CRC samples (combining intensity score 0-3 and density score 0-4)

• Verifying specificity:

  • Problem: Uncertainty about antibody specificity

  • Solution: Include appropriate positive controls (testis tissue has shown positive SOX30 staining) and negative controls (liver and skin tissues have shown negative staining)

  • Additional validation can include correlation with Western blot results from the same samples

• Low signal in tissues with reduced SOX30 expression:

  • Problem: Detection challenges in cancer tissues with downregulated SOX30

  • Solution: Use amplification systems; consider more sensitive detection methods; optimize counterstaining to enhance contrast

These troubleshooting approaches can improve the reliability and consistency of SOX30 detection in tissue samples.

How can researchers validate SOX30 antibody specificity for their experimental systems?

Thorough validation of SOX30 antibody specificity is crucial for experimental reliability. Researchers should implement these comprehensive validation strategies:

• Positive and negative controls:

  • Utilize SOX30-overexpressing cell lines (e.g., SOX30-transfected HEK293T cells) as positive controls

  • Include vector-only transfected cells as negative controls

  • Use tissues with known SOX30 expression patterns (positive: testis; negative: liver, skin)

• siRNA/shRNA knockdown validation:

  • Perform SOX30 knockdown experiments using specific siRNA/shRNA

  • Confirm reduced signal with SOX30 antibody following knockdown

  • This orthogonal approach provides strong evidence of antibody specificity

• Multi-technique confirmation:

  • Compare results across multiple detection methods (IHC, Western blot, immunofluorescence)

  • Verify correlation between techniques (as demonstrated in CRC studies where SOX30 detected by Western blot was highly correlated with SOX30 detected by IHC)

• Peptide competition assay:

  • Pre-incubate SOX30 antibody with excess immunizing peptide

  • Demonstrate abolishment of specific staining in IHC or bands in Western blot

• Recombinant protein testing:

  • Test antibody against purified recombinant SOX30 protein

  • Verify detection of protein at the expected molecular weight (82 kDa)

• Cross-reactivity assessment:

  • Test antibody against related SOX family proteins to confirm specificity

  • Particularly important due to conserved domains within the SOX family

• Independent antibody comparison:

  • Compare results using different antibodies targeting distinct epitopes of SOX30

  • Consistent results across different antibodies increase confidence in specificity

Implementing these validation strategies ensures reliable and reproducible results in SOX30 research.

How can SOX30 antibodies be utilized in studying the molecular mechanisms of cancer suppression?

SOX30 antibodies provide powerful tools for investigating the molecular mechanisms underlying SOX30's tumor-suppressive functions through these advanced research applications:

• Chromatin Immunoprecipitation (ChIP) studies:

  • Use SOX30 antibodies to identify direct genomic binding sites, particularly focusing on:

    • The p53 promoter region, where SOX30 directly binds to CACTTTG (+115 to +121) to activate p53 transcription

    • The CTNNB1 promoter, where SOX30 binds to repress transcription and inhibit Wnt signaling

  • Combine with sequencing (ChIP-seq) for genome-wide binding profiles of SOX30

• Protein-protein interaction studies:

  • Employ co-immunoprecipitation (co-IP) with SOX30 antibodies to identify binding partners

  • Investigate SOX30 interaction with CTNNB1 protein, which prevents CTNNB1 interaction with TCF7L2/TCF4

  • Map interaction domains using deletion constructs and co-IP

• Pathway analysis:

  • Use SOX30 antibodies in combination with antibodies against:

    • Wnt signaling components to elucidate SOX30's role in pathway inhibition

    • p53 pathway proteins to confirm transcriptional activation effects

    • EMT markers to understand SOX30's role in suppressing metastasis

• Loss and gain of function studies:

  • Validate signaling changes following SOX30 manipulation using antibody-based detection methods

  • Monitor downstream effects of SOX30 overexpression or knockdown on:

    • Cancer cell apoptosis and proliferation

    • p53 expression and activation

    • Wnt signaling activity

• In vivo tumor models:

  • Use SOX30 antibodies for IHC analysis of tumor xenografts with manipulated SOX30 expression

  • Correlate SOX30 expression with tumor growth rates, metastasis, and survival outcomes

• Epigenetic regulation mechanisms:

  • Combine demethylation studies with antibody-based detection to confirm restoration of SOX30 expression

  • Investigate how epigenetic silencing of SOX30 through hypermethylation contributes to tumorigenesis

These advanced applications allow researchers to comprehensively characterize SOX30's role in cancer suppression mechanisms.

What approaches can researchers use to study SOX30 in relation to therapeutic response?

Researchers can employ several sophisticated approaches to investigate SOX30's role in therapeutic response:

• Patient-derived xenograft (PDX) models:

  • Establish PDX models with varying SOX30 expression levels

  • Use SOX30 antibodies to characterize expression in PDX samples

  • Correlate SOX30 expression with response to standard chemotherapies and targeted agents

  • Based on findings that SOX30 may decrease chemoresistance by promoting p53 transcriptional activation

• Cell line sensitivity profiling:

  • Generate isogenic cell lines with SOX30 overexpression or knockdown

  • Perform drug sensitivity screens to identify therapeutic vulnerabilities

  • Use SOX30 antibodies to confirm expression levels and correlate with drug response

  • Focus on p53-activating compounds, given SOX30's role in p53 regulation

• Combinatorial therapy assessment:

  • Investigate whether epigenetic modifying drugs that reverse SOX30 methylation can sensitize cancer cells to conventional therapies

  • Test combinations of demethylating agents with chemotherapeutics

  • Monitor SOX30 re-expression using antibody-based detection methods

• Mechanistic response studies:

  • Use SOX30 antibodies in time-course experiments following drug treatment

  • Monitor changes in SOX30 localization, expression, or post-translational modifications

  • Combine with analysis of downstream pathway components (p53, Wnt signaling elements)

• Predictive biomarker development:

  • Perform retrospective analysis of SOX30 expression in patient samples with known treatment outcomes

  • Correlate SOX30 levels (detected by IHC) with response to specific therapeutic regimens

  • Develop standardized scoring systems for potential clinical application

• Resistance mechanism investigation:

  • Study SOX30 expression changes in cell lines with acquired drug resistance

  • Determine whether SOX30 downregulation contributes to resistance development

  • Test whether restoring SOX30 expression can re-sensitize resistant cells

These approaches can provide valuable insights into SOX30's potential as a predictive biomarker and therapeutic target in cancer treatment.

How might SOX30 be integrated into precision oncology approaches?

The integration of SOX30 into precision oncology frameworks represents an emerging research frontier with several promising avenues:

• Prognostic stratification refinement:

  • Develop standardized SOX30 IHC scoring systems for clinical implementation

  • Create integrated prognostic models combining SOX30 with established biomarkers

  • The demonstrated independent prognostic value of SOX30 in colorectal cancer suggests potential utility in refining risk stratification

• Predictive biomarker development:

  • Investigate SOX30 expression as a predictor of response to:

    • Conventional chemotherapies, leveraging SOX30's role in decreasing chemoresistance

    • Wnt pathway inhibitors, based on SOX30's function in Wnt signaling regulation

    • p53-activating compounds, given SOX30's direct regulation of p53 transcription

• Epigenetic therapy opportunities:

  • Explore targeted demethylation approaches to restore SOX30 expression in cancers with SOX30 hypermethylation

  • Design screening assays for compounds that specifically reactivate SOX30 expression

  • Investigate combinatorial approaches with conventional therapies

• Liquid biopsy applications:

  • Develop methods to detect SOX30 methylation in circulating tumor DNA

  • Monitor SOX30 methylation status as a potential biomarker for early detection or disease monitoring

  • Based on the high frequency of SOX30 hypermethylation observed in lung cancer (70.83% of primary tumors)

• Pathway-directed therapeutic strategies:

  • Design therapeutic approaches targeting the specific pathways regulated by SOX30 (Wnt, p53)

  • Develop synthetic lethality strategies for tumors with SOX30 loss

• Multi-omic integration:

  • Combine SOX30 protein expression data with genomic, transcriptomic, and epigenomic profiles

  • Generate comprehensive predictive models for personalized treatment selection

  • Identify context-dependent functions of SOX30 across different tumor types

The integration of SOX30 into precision oncology approaches holds significant potential for improving patient stratification and treatment selection across multiple cancer types.

What are the current limitations in SOX30 research and how might they be addressed?

Despite significant advances, several limitations exist in SOX30 research that require methodological innovations:

• Tissue-specific expression heterogeneity:

  • Limitation: Variable SOX30 expression across tissues complicates interpretation

  • Solution: Develop single-cell analysis methods incorporating SOX30 antibodies to map expression at cellular resolution

  • Implement spatial transcriptomics approaches to correlate SOX30 protein expression with cellular context

• Post-translational modification characterization:

  • Limitation: Limited understanding of how PTMs regulate SOX30 function

  • Solution: Develop modification-specific antibodies (phospho-SOX30, acetyl-SOX30)

  • Apply mass spectrometry approaches to comprehensively map SOX30 modifications

• Mechanistic pathway understanding:

  • Limitation: Incomplete knowledge of the full spectrum of SOX30 target genes

  • Solution: Combine ChIP-seq with RNA-seq following SOX30 manipulation

  • Develop inducible SOX30 expression systems for temporal analysis of transcriptional networks

• Animal models:

  • Limitation: Few established animal models for SOX30 functional studies

  • Solution: Generate conditional SOX30 knockout/knockin mouse models

  • Develop tissue-specific SOX30 expression systems to study developmental and pathological roles

• Technical antibody limitations:

  • Limitation: Current antibodies may not recognize all SOX30 isoforms or post-translationally modified forms

  • Solution: Develop isoform-specific antibodies

  • Generate antibodies against different epitopes to ensure comprehensive detection

• Translation to clinical applications:

  • Limitation: Standardization challenges for SOX30 detection in clinical samples

  • Solution: Establish multi-institutional protocols for SOX30 IHC

  • Develop automated scoring systems for consistent quantification

• Functional redundancy with other SOX family members:

  • Limitation: Potential compensatory mechanisms complicating interpretation

  • Solution: Study SOX30 in context with related SOX family members

  • Develop multiplexed detection approaches to simultaneously analyze multiple SOX proteins

Addressing these limitations through methodological innovations will advance our understanding of SOX30 biology and its potential applications in cancer research and therapy.