SOX9 Antibody, HRP conjugated

What is SOX9 and why is it significant in research?

SOX9 belongs to the SOX (SRY-like HMG box) family of transcription factors with diverse roles in development. It is expressed in mesenchymal progenitors that give rise to chondrocytes and osteoblasts, as well as in the central nervous system, neural crest, intestine, pancreas, and testis. Mutations in SOX9 are associated with defects in sex determination, cartilage and bone development, and abnormalities of the heart, kidneys, brain, gut, and pancreas . SOX9's critical role in multiple developmental processes makes it an important target for understanding tissue differentiation, organ development, and disease pathogenesis.

What are the primary applications for SOX9 antibodies in research?

SOX9 antibodies are utilized across multiple experimental platforms:

• Western blotting for protein expression analysis in cell and tissue lysates

• Immunocytochemistry/Immunofluorescence for cellular and subcellular localization

• Direct ELISA for quantitative detection and antibody validation

• Immunohistochemistry for tissue-specific expression patterns

• Flow cytometry for identifying SOX9-expressing cell populations

These applications enable researchers to investigate SOX9 expression, regulation, and function in various biological contexts ranging from normal development to disease states.

How do HRP-conjugated SOX9 antibodies differ from non-conjugated versions?

HRP-conjugated SOX9 antibodies have the horseradish peroxidase enzyme directly attached to the antibody that recognizes SOX9. This conjugation eliminates the need for a secondary antibody step, streamlining detection protocols and potentially reducing background signal. In contrast, non-conjugated antibodies require a secondary antibody (often HRP-conjugated) to bind to the primary antibody for detection. The search results show examples where non-conjugated primary antibodies were used with HRP-conjugated secondary antibodies for detection in Western blots and immunohistochemistry applications .

What are the optimal conditions for Western blot detection of SOX9?

For optimal Western blot detection of SOX9, the following conditions have been validated:

A specific band for SOX9 is typically detected at approximately 75 kDa, with GAPDH often used as a loading control .

How should I optimize immunocytochemistry protocols for SOX9 detection?

For effective immunocytochemistry/immunofluorescence detection of SOX9:

• Use immersion fixation for cells

• Apply SOX9 antibody at 10 μg/mL concentration

• Incubate for 3 hours at room temperature

• Use appropriate fluorophore-conjugated secondary antibody (e.g., NorthernLights 557-conjugated Anti-Goat IgG)

• Counterstain with DAPI to visualize nuclei

• Expect nuclear localization of SOX9 staining

In embryonic stem cells and progenitor cells, SOX9 shows specific nuclear localization that can be effectively visualized using these protocols .

What cell and tissue types have been validated for SOX9 antibody applications?

SOX9 antibodies have been validated in diverse biological specimens:

These validated systems provide reference points for researchers using SOX9 antibodies in similar or novel contexts .

How can I address cross-reactivity issues with SOX9 antibodies?

SOX9 antibodies may exhibit cross-reactivity with related proteins, particularly other SOX family members. The search results indicate approximately 25% cross-reactivity with recombinant human SOX10 in direct ELISAs for some antibodies . To minimize cross-reactivity concerns:

• Select antibodies with validated specificity through multiple techniques

• Include appropriate positive and negative controls

• Validate specificity in your specific experimental system

• Consider using multiple antibodies targeting different SOX9 epitopes

• Perform validation experiments such as antibody neutralization

• Verify results with complementary techniques (e.g., qPCR)

These strategies help ensure that observed signals are specific to SOX9 rather than related proteins.

What are common challenges in detecting SOX9 in different cell types?

Detecting SOX9 presents several technical challenges:

• Variable expression levels: SOX9 expression can vary significantly between cell types and developmental stages

• Nuclear localization: As a transcription factor, SOX9 localizes primarily to nuclei, requiring effective nuclear extraction protocols

• Post-translational modifications: SOX9 undergoes modifications that affect molecular weight and antibody recognition

• Molecular weight variations: SOX9 appears at different molecular weights (56-107 kDa) depending on context

• Background signal: Non-specific binding can occur, especially in immunohistochemistry

To address these challenges, optimize protocols for each specific cell type and application, and include appropriate controls to validate specificity of detection .

How can I validate SOX9 antibody specificity in my experiments?

To ensure SOX9 antibody specificity:

• Use multiple antibodies targeting different epitopes of SOX9

• Include proper positive controls (cells/tissues known to express SOX9) and negative controls

• Perform knockdown experiments to confirm signal reduction

• Compare staining patterns with published SOX9 expression patterns

• Conduct peptide competition assays

• Verify results using complementary techniques (e.g., Western blot, qPCR)

• Check for expected subcellular localization (primarily nuclear)

The search results demonstrate validation approaches including detection of SOX9 in multiple cell lines and consistent nuclear staining patterns .

How can SOX9 antibodies help investigate TGF-β signaling interactions?

SOX9 antibodies can reveal important interactions between SOX9 and the TGF-β pathway:

The search results demonstrate that TGF-β1 treatment stabilizes SOX9 protein without affecting SOX9 mRNA levels in bovine chondrocytes. Western blot analysis using SOX9 antibodies showed increased SOX9 protein levels after 4-6 hours of TGF-β1 treatment despite unchanged mRNA expression. This was accompanied by increased phosphorylated SMAD3, confirming activation of TGF-β signaling .

To investigate these interactions:

• Design time-course experiments with TGF-β treatment

• Use SOX9 antibodies alongside phospho-SMAD3 antibodies

• Examine correlations between pathway activation and SOX9 stability

• Explore downstream effects on SOX9 target genes like PAPSS2

This approach helps elucidate post-transcriptional regulation mechanisms of SOX9.

What insights can SOX9 antibodies provide in cancer research?

SOX9 antibodies offer valuable insights into cancer biology:

The search results show SOX9 expression in multiple cancer cell lines including HeLa (cervical), KATO-III (gastric), COLO 205 (colorectal), and Hep3B (hepatocellular) . Additionally, immunohistochemistry demonstrates SOX9 expression in colorectal adenocarcinoma and liver cancer tissues .

Particularly interesting is the finding that HGF stimulation enhances SOX9 expression in cancer cells while simultaneously regulating cancer stem cell markers (CD49b, CD49f, CD44, CD24). Western blot analysis showed enhancement of SOX9 expression upon HGF stimulation from 2 to 24 hours, suggesting SOX9 may play a role in cancer stem cell properties .

These applications enable researchers to investigate SOX9's role in tumorigenesis, cancer stem cells, and potential therapeutic interventions.

How can SOX9 antibodies be used in developmental biology and stem cell research?

SOX9 antibodies are powerful tools for developmental and stem cell research:

The search results demonstrate SOX9 detection in embryonic stem cells differentiated into early proximal lung progenitor cells, showing nuclear localization . Additionally, SOX9+ basal cells (BCs) in human airways have been characterized using SOX9 antibodies in combination with other markers (P63, KRT5, CC10, KI67) .

SOX9 antibodies can be used to:

• Track lineage specification during differentiation protocols

• Identify SOX9+ progenitor populations in developing tissues

• Monitor SOX9 expression through early and late passages of cultured progenitor cells

• Analyze co-expression with other developmental markers

• Validate gene expression studies at the protein level

qPCR and Western blotting with SOX9 antibodies have been used to compare progenitor cell marker expression between human lung samples and cultured SOX9+ basal cells through early (P2) and late (P8) passages .

How should I interpret variations in SOX9 molecular weight?

SOX9 shows notable molecular weight variations across experimental systems:

These variations likely reflect:

• Post-translational modifications (phosphorylation, SUMOylation)

• Tissue/cell-specific processing

• Experimental conditions affecting protein migration

• Potential protein complex formation

When interpreting results, researchers should consider these factors and validate findings using appropriate controls .

What considerations are important when comparing SOX9 protein levels across experimental conditions?

When comparing SOX9 protein levels:

• Loading controls: Use appropriate housekeeping proteins (e.g., GAPDH) for normalization

• Antibody consistency: Maintain consistent antibody concentrations across experiments

• Detection parameters: Use identical exposure times and imaging settings

• Biological replication: Include sufficient biological replicates (n=3 minimum)

• Statistical analysis: Apply appropriate statistical tests for significance

• Sample handling: Control for variations in extraction efficiency, especially for nuclear proteins

The search results demonstrate proper use of loading controls (GAPDH) in Western blots and adequate biological replication (n=3) in qPCR analyses .

How can I analyze SOX9 expression in heterogeneous tissues?

For analyzing SOX9 in complex tissues:

• Use multiplexed immunofluorescence with SOX9 and other markers

• Perform co-localization studies to identify specific cell populations

• Consider sequential sections when antibody compatibility is an issue

• Apply digital image analysis for quantitative assessment

• Validate findings with multiple techniques (e.g., FACS, qPCR)

The search results show co-staining of SOX9 with markers like P63, KRT5, and CC10 in airway epithelium, and flow cytometry analysis of SOX9 expression in relation to CD44/CD24 populations in cancer cells . These approaches can reveal cell-type specific expression patterns and regulatory relationships in heterogeneous tissues.