SOX21 Antibody

What is SOX21 and why is it important in research?

SOX21 (SRY-box transcription factor 21) is a nuclear protein of approximately 28.6 kDa with 276 amino acid residues in humans. It functions primarily as a transcription factor, playing crucial roles in neural development, epithelial cell differentiation, and as an activator of OPRM1 transcription. SOX21 is also implicated in ameloblast differentiation and has been studied in the context of neurogenesis and pulmonary neuroendocrine cell development . Understanding SOX21 function is important for developmental biology, neuroscience, and cancer research as it regulates important developmental pathways.

How do I select the appropriate SOX21 antibody for my research?

When selecting a SOX21 antibody, consider the following factors:

• Application compatibility: Determine if the antibody has been validated for your specific application (WB, IHC, ICC/IF, ELISA)

• Species reactivity: Verify that the antibody recognizes SOX21 in your species of interest

• Epitope specificity: Review if the antibody targets a unique region of SOX21 to avoid cross-reactivity

• Validation data: Examine available validation data including images showing specificity

• Clonality: Choose between monoclonal (higher specificity) or polyclonal (broader epitope recognition) based on research needs

For example, if detecting SOX21 in human and mouse samples via Western blot and immunocytochemistry, antibodies like those from Abcam (ab220024, ab220025) offer validated reactivity for both species and multiple applications .

What are the common synonyms and identifiers for SOX21?

When searching literature or databases for SOX21-related information, be aware of these alternative identifiers:

These identifiers are particularly important when searching literature databases or ordering specific antibodies for research applications .

What are the optimal conditions for Western blot detection of SOX21?

For successful Western blot detection of SOX21:

• Sample preparation: Nuclear extraction methods are recommended as SOX21 is primarily localized in the nucleus

• Expected band size: Look for bands between 25-30 kDa (the observed molecular weight can vary slightly from the calculated 28.6 kDa)

• Dilution ranges:

  • Most commercial antibodies work optimally at 1:200-1:1000 dilution for WB

  • For Proteintech's antibody (55052-1-AP), a dilution of 1:200-1:1000 is recommended

  • R&D Systems' antibody works at 0.1 μg/mL for WB

• Positive controls: Jurkat cells and mouse embryo tissue have shown positive detection

• Blocking: 5% non-fat milk in TBST is typically sufficient

Always validate the specificity with appropriate controls, as SOX21 belongs to the SOX family with high homology among members .

How can I optimize immunohistochemistry for SOX21 detection in tissue sections?

For optimal IHC detection of SOX21:

• Epitope retrieval: Heat-induced epitope retrieval using basic pH buffer (such as VisUCyte Antigen Retrieval Reagent-Basic) is recommended for optimal antigen exposure

• Antibody concentration:

  • For paraffin-embedded sections, 0.5 μg/ml concentration is typically effective

  • Incubation time of 1 hour at room temperature is standard

• Detection systems:

  • HRP-conjugated secondary antibodies or polymer-based detection systems both work well

  • DAB (3,3'-diaminobenzidine) as chromogen with hematoxylin counterstain shows good contrast

• Positive controls: Human glioblastoma tissue shows specific nuclear staining for SOX21

• Expected pattern: Look for specific nuclear localization, as SOX21 is a transcription factor

Researchers should optimize these conditions based on their specific tissue type and fixation method.

What are the validated applications for SOX21 antibodies and their specific protocols?

SOX21 antibodies have been validated for multiple applications:

When switching between applications, optimization of antibody concentration is necessary to achieve specific signal with minimal background .

How can I distinguish between SOX21 and other SOX family members in my experiments?

The SOX family shares significant homology, making specific detection challenging. To ensure SOX21 specificity:

• Epitope selection: Choose antibodies raised against unique regions of SOX21 not conserved in other SOX proteins

• Cross-reactivity testing: R&D Systems antibody shows <1% cross-reactivity with SOX1, SOX2, and SOX15 in direct ELISAs and Western blots

• Validation approaches:

  • Knockout/knockdown controls: Use SOX21 knockout/knockdown samples as negative controls

  • Recombinant protein competition: Pre-incubate antibody with recombinant SOX21 to confirm specificity

  • Multiple antibody validation: Use antibodies targeting different epitopes of SOX21

• Sequence alignment: Run a bioinformatic analysis of your antibody's epitope against other SOX proteins to predict potential cross-reactivity

The high homology in the HMG-box DNA-binding domain is particularly challenging, so antibodies targeting the C-terminal region (e.g., Abcam's antibodies targeting aa 250 to C-terminus) typically offer better specificity .

What are the common issues when working with SOX21 antibodies and how can they be resolved?

Researchers commonly encounter these challenges with SOX21 antibodies:

When troubleshooting, always include appropriate positive and negative controls to validate your experimental conditions .

How can I use SOX21 antibodies to study its role in neurogenesis and neural development?

To investigate SOX21's role in neurogenesis:

• Temporal expression analysis: Use Western blot and IHC to track SOX21 expression during different stages of neural development

• Co-localization studies:

  • Perform double immunofluorescence with SOX21 antibody and neural progenitor markers (Nestin, Sox2)

  • Use confocal microscopy to determine precise cellular localization

• Functional studies:

  • Combine SOX21 immunostaining with BrdU labeling to identify proliferating neural progenitors

  • Correlate SOX21 expression with differentiation markers in gain/loss-of-function models

• Transcriptional regulation:

  • Use ChIP assays with SOX21 antibodies to identify direct target genes

  • Focus on the Hes5 gene, which has been identified as a target for SOX21-mediated transcriptional repression in hippocampal adult neurogenesis

Research has shown that SOX21 promotes hippocampal adult neurogenesis through transcriptional repression of the Hes5 gene, making this a key pathway to investigate in neural development studies .

What are the expected expression patterns of SOX21 in different tissues and cell types?

SOX21 shows distinct expression patterns across tissues:

When interpreting results, compare your findings with these established patterns and consider developmental stage and pathological state which can significantly alter expression levels .

How does SOX21 interact with other SOX family members, particularly SOX2?

SOX21 has complex interactions with other SOX family members:

• SOX2-SOX21 interaction in neural development:

  • SOX21 often counteracts SOX2 activity in neural progenitor differentiation

  • While SOX2 maintains stemness, SOX21 can promote differentiation

• SOX2-SOX21 interaction in epithelial cells:

  • In extrapulmonary airways, SOX21 modulates SOX2-initiated differentiation

  • Their balance affects cell fate decisions in epithelial development

• Regulatory network:

  • SOX21 can function as a transcriptional repressor for some SOX2 target genes

  • They may compete for binding to similar DNA motifs due to their conserved HMG domains

• Co-expression patterns:

  • In some developmental contexts, they show overlapping expression

  • In others, they show mutually exclusive expression patterns

When designing experiments to study these interactions, consider using co-immunoprecipitation with SOX21 antibodies to identify protein-protein interactions, or ChIP-seq to identify overlapping and distinct genomic targets .

What is known about SOX21's role in pathological conditions and can antibodies help study these connections?

SOX21 has been implicated in several pathological conditions:

• Cancer research applications:

  • SOX21 antibodies have been used to study expression in glioblastoma samples

  • Immunohistochemistry shows nuclear localization in tumor tissues

  • Changes in SOX21 expression may correlate with tumor progression

• Neurodevelopmental disorders:

  • Given SOX21's role in neurogenesis, antibodies can help study its expression in models of neurodevelopmental disorders

  • Alterations in SOX21 levels may contribute to neurological pathologies

• Epithelial disorders:

  • SOX21 modulates epithelial cell differentiation in airways

  • Antibodies can detect abnormal expression in respiratory epithelial disorders

• Research approach:

  • Use SOX21 antibodies for tissue microarray analysis to compare expression across multiple patient samples

  • Correlate expression levels with clinical outcomes through quantitative image analysis of immunostained sections

Recent research has identified a potential role for SOX21 in colon cancer, where it may influence cell growth and metastasis through Hedgehog signaling axis, highlighting the importance of studying SOX21 in various pathological contexts .

How can I quantify SOX21 expression levels in experimental samples?

For accurate quantification of SOX21 expression:

• Western blot quantification:

  • Use appropriate loading controls (nuclear proteins like Lamin B1 are preferable since SOX21 is nuclear)

  • Employ densitometry software (ImageJ, Image Lab) to measure band intensity

  • Normalize SOX21 signal to loading control for relative quantification

• Immunofluorescence quantification:

  • Measure nuclear fluorescence intensity using software like CellProfiler or ImageJ

  • Quantify percentage of SOX21-positive cells in different conditions

  • Use nuclear counterstain (DAPI) for normalization and cell counting

• qPCR complementation:

  • Complement protein-level data with mRNA quantification

  • Design specific primers spanning exon-exon junctions for specificity

• Considerations for accuracy:

  • Include calibration curves with recombinant SOX21 for absolute quantification

  • Use the same antibody lot and consistent exposure times between experiments

  • Include biological replicates and appropriate statistical analysis

For relative quantification across experimental conditions, consistent methodology and appropriate normalization are essential for reliable results .

How can I use SOX21 antibodies in ChIP (Chromatin Immunoprecipitation) experiments?

For effective ChIP experiments with SOX21 antibodies:

• Antibody selection: Choose ChIP-grade antibodies specifically validated for immunoprecipitation, as not all SOX21 antibodies work efficiently for ChIP

• Protocol optimization:

  • Cross-linking: 1% formaldehyde for 10 minutes at room temperature is standard

  • Sonication: Optimize to achieve 200-500 bp DNA fragments

  • Immunoprecipitation: Use 2-5 μg of SOX21 antibody per ChIP reaction

  • Controls: Include IgG negative control and positive control for a known SOX21 target

• Target validation:

  • Design primers for OPRM1 promoter regions, a known SOX21 target

  • The Hes5 gene promoter is another validated target for SOX21 binding

• Data analysis:

  • Quantify enrichment by qPCR comparing to input and IgG control

  • For genome-wide binding, consider ChIP-seq approach with bioinformatic analysis

This approach can reveal direct transcriptional targets of SOX21 and help understand its regulatory network in your biological system of interest .

What are the considerations for using SOX21 antibodies in flow cytometry applications?

When using SOX21 antibodies for flow cytometry:

• Sample preparation challenges:

  • As a nuclear transcription factor, SOX21 requires effective fixation and permeabilization

  • Use 4% paraformaldehyde fixation followed by permeabilization with 0.1-0.5% Triton X-100

• Antibody selection:

  • Choose unconjugated antibodies and use appropriate fluorophore-conjugated secondary antibodies

  • Alternatively, select directly conjugated antibodies if available, though these are less common for SOX21

• Protocol considerations:

  • Include RNase treatment to reduce background

  • Longer primary antibody incubation (overnight at 4°C) may improve signal

  • Use transcription factor staining buffers specifically designed for nuclear proteins

• Controls and validation:

  • Include isotype controls at the same concentration as the primary antibody

  • Use cell lines with known SOX21 expression (positive) and knockdown/knockout cells (negative) to validate specificity

  • Consider co-staining with other markers to identify specific cell populations

• Data analysis:

  • Analyze shifts in fluorescence intensity compared to controls

  • Consider using median fluorescence intensity rather than mean for more robust quantification

While flow cytometry is less commonly used for SOX21 than imaging techniques, it offers the advantage of quantifying expression across large cell populations .

How can I design experiments to study SOX21's functional role in gene regulation?

To investigate SOX21's functional role in gene regulation:

• Gene expression correlation studies:

  • Use SOX21 antibodies for immunoprecipitation followed by mass spectrometry to identify protein interaction partners

  • Perform Western blot analysis of SOX21 alongside potential target genes in various conditions

• Knockdown/overexpression approaches:

  • Create SOX21 knockdown/knockout models using siRNA or CRISPR-Cas9

  • Generate SOX21 overexpression systems using appropriate expression vectors

  • In both cases, validate SOX21 protein levels using validated antibodies before assessing downstream effects

• Reporter gene assays:

  • Design luciferase reporters containing promoters of suspected SOX21 target genes (e.g., OPRM1 or Hes5)

  • Co-transfect with SOX21 expression constructs to measure transcriptional activation or repression

• ChIP-seq and RNA-seq integration:

  • Perform ChIP-seq using SOX21 antibodies to identify genome-wide binding sites

  • Complement with RNA-seq after SOX21 manipulation to correlate binding with expression changes

  • Integrate these datasets to identify direct regulatory targets

Research has shown that SOX21 can upregulate OPRM1 distal promoter activity in mor-expressing neuronal cells, demonstrating its role as a transcriptional activator in specific contexts .