SOX3 Antibody, FITC conjugated

What is SOX3 and what are its primary biological functions?

SOX3 is a transcription factor that plays crucial roles in multiple developmental processes. It is required during the formation of the hypothalamo-pituitary axis and serves as a developmental switch in neuronal development. SOX3 maintains neural cells in an undifferentiated state by counteracting the activity of proneural proteins, effectively suppressing neuronal differentiation. Beyond neural development, SOX3 is required within pharyngeal epithelia for proper craniofacial morphogenesis. In sex determination, SOX3 controls genetic switches in male development by directing the development of supporting cell precursors (pre-Sertoli cells) into Sertoli rather than granulosa cells . Additionally, SOX3 functions as an ancestral homologous gene to the male-determining Sry in eutherian mammals and determines maleness in medaka fish .

What are the key specifications of the SOX3 Polyclonal Antibody, FITC Conjugated?

The SOX3 Polyclonal Antibody with FITC conjugation has the following specifications:

This antibody is designed for applications such as ELISA, with recommended dilutions ranging from 1:100 to 1:500 for optimal results .

How should researchers validate the specificity of SOX3 antibodies in their experimental systems?

Validating antibody specificity is critical for reliable results. Researchers should implement multiple validation approaches: (1) Compare immunostaining between wild-type tissues and SOX3-null or knockout tissues - this provides definitive evidence of specificity as demonstrated in studies comparing SOX3 immunostaining on Sox3-null cells (from 14.5 dpc +/− embryos) and Sox3-expressing cells . (2) Perform Western blots using positive control samples (e.g., NTera-2 human testicular embryonic carcinoma cell line) alongside negative controls (e.g., HDLM-2 human Hodgkin's lymphoma cells) as documented in published validations . (3) Conduct parallel analyses using alternative SOX3 detection methods such as in situ hybridization (ISH) to confirm protein expression patterns correspond with transcript localization. (4) Include additional controls where SOX3 is experimentally manipulated, such as comparing protein levels between wild-type cells and cells with SOX3 mutations (e.g., Sox3-26ala mutants) where transcription remains intact but protein levels are altered .

What are the optimal sample preparation protocols for detecting SOX3 in different cellular compartments?

Sample preparation protocols must be tailored to SOX3's predominantly nuclear localization as a transcription factor. For immunofluorescence detection in cultured cells: (1) Fix cells using freshly prepared 4% paraformaldehyde for 15-20 minutes at room temperature to preserve nuclear architecture. (2) Use a permeabilization buffer containing 0.2-0.5% Triton X-100 to facilitate antibody access to nuclear SOX3. (3) Block with 5-10% normal serum from the species of the secondary antibody for at least 1 hour. (4) Apply the SOX3-FITC conjugated antibody at 10-15 μg/mL overnight at 4°C or for 3 hours at room temperature as demonstrated in protocols used for NTera-2 and U-251 MG cells . (5) For tissue sections, antigen retrieval is critical - researchers successfully detected SOX3 in telencephalic ventricular zone cells using heat-induced epitope retrieval in citrate buffer (pH 6.0) . For biochemical analyses requiring nuclear protein isolation, implement differential centrifugation protocols that effectively separate nuclear from cytoplasmic compartments, as verified by nuclear markers (e.g., Histone H3) and absence of cytoplasmic contamination (e.g., α-Tubulin negativity) .

How can SOX3-FITC antibody be effectively used to study neural development processes?

For neural development studies, SOX3-FITC antibody applications should focus on neural progenitor populations where SOX3 functions as a critical regulator. Researchers should: (1) Establish developmental time series experiments tracking SOX3 expression in neural progenitors, particularly in the ventricular zone where SOX3 protein is present in all wild-type cells at 13.5 dpc in mouse telencephalon . (2) Combine SOX3-FITC antibody with markers of cell proliferation (e.g., Ki67) to distinguish between proliferative and differentiating neural populations. (3) Implement co-staining with neuronal differentiation markers (e.g., βIII-tubulin) to visualize the transition from SOX3+ progenitors to differentiated neurons. (4) For in vitro models, apply the antibody to neural differentiation systems such as embryonic stem cells grown in N2B27 medium for 4 days to form neural progenitors, where SOX3 serves as a key marker of neural commitment . (5) Utilize confocal microscopy with z-stack acquisition to precisely locate SOX3 within the nuclear compartment and determine co-localization with other transcription factors involved in neurogenesis.

What controls are essential when designing SOX3 knockdown or knockout experiments?

When designing SOX3 knockdown or knockout experiments, several critical controls must be implemented: (1) Include parallel analysis of SOX3 at both protein and transcript levels - as demonstrated in Sox3-26ala mutants, transcript levels can remain unchanged while protein levels are dramatically reduced, requiring qPCR verification alongside Western blotting or immunostaining . (2) Utilize multiple knockout/knockdown strategies targeting different regions of the SOX3 gene - for example, TALEN constructs targeting three distinct sequences (between ATG and HMG domain, and within the transcription activation domain) produced different phenotypic outcomes in frog experiments . (3) Include genotyping controls to confirm genetic modifications, as implemented in studies using primer sets that amplify fragments containing TALEN target sites . (4) Establish dose-dependency by comparing heterozygous and homozygous knockouts - significant differences in follicle development were observed between sox3+/+ and sox3−/− zebrafish . (5) Perform rescue experiments by re-introducing wild-type SOX3 to confirm phenotype specificity, particularly when studying sex reversal or reproductive phenotypes .

How should researchers interpret conflicting SOX3 protein and mRNA expression data?

When faced with discrepancies between SOX3 protein detection and mRNA expression data, researchers should consider several explanations and troubleshooting approaches: (1) Post-transcriptional regulation - as observed in Sox3-26ala mutants where transcription is unaffected but protein is cleared from cells, demonstrating that normal transcript levels don't guarantee corresponding protein levels . (2) Protein stability differences - SOX3 protein may undergo rapid turnover in certain cellular contexts, requiring proteasome inhibitors to stabilize expression for detection. (3) Technical limitations in antibody sensitivity - researchers should consider exposure times as demonstrated in studies comparing 3-minute versus 30-minute Western blot exposures to detect low levels of SOX3 protein . (4) Subcellular localization changes - SOX3 might relocalize within cells under experimental conditions while maintaining transcript levels, necessitating fractionation approaches. (5) For reconciling these differences, implement parallel in situ hybridization and immunohistochemistry on adjacent tissue sections (10 μm apart) as performed on 13.5 dpc chimeric telencephalon to directly compare transcript and protein distribution patterns . (6) Quantify protein degradation rates through cycloheximide chase experiments to determine if protein stability rather than transcription explains observed differences.

What are the most common technical challenges when using SOX3-FITC antibodies in immunohistochemistry and how can they be overcome?

Common technical challenges with SOX3-FITC antibodies in immunohistochemistry include: (1) Background autofluorescence - minimize by using freshly prepared paraformaldehyde for fixation and including an autofluorescence quenching step with 0.1% sodium borohydride. (2) Signal-to-noise ratio issues - improve by optimizing antibody concentration through titration experiments; published protocols suggest 10-15 μg/mL for cell lines . (3) Epitope masking - conduct systematic comparison of antigen retrieval methods, as SOX3 detection in tissue sections typically requires heat-induced epitope retrieval. (4) Photobleaching of FITC conjugate - minimize exposure to light during all procedures and consider using anti-fade mounting media containing propyl gallate or p-phenylenediamine. (5) Signal specificity concerns - include multiple controls including SOX3-null tissues alongside experimental samples in the same slides, as demonstrated in studies comparing Sox3-null and Sox3-26ala expressing cells . (6) Variability between tissue types - establish tissue-specific optimization protocols, as fixation times may need adjustment between highly cellular neural tissues versus gonadal tissues. (7) For multiplexing with other antibodies, consider the order of application and potential steric hindrance issues, particularly when targeting multiple nuclear transcription factors.

How can researchers differentiate between specific SOX3 signals and potential cross-reactivity with other SOX family members?

Differentiating specific SOX3 signals from cross-reactivity with other SOX family proteins requires rigorous validation: (1) Carefully examine the immunogen sequence (4-118AA of human SOX3) and compare with homologous regions in other SOX proteins, particularly SOX1 and SOX2 which share high sequence similarity. (2) Perform parallel experiments in tissues with known differential expression of SOX family members, as demonstrated in studies comparing U-251 MG glioblastoma cells (SOX3-positive) versus HDLM-2 Hodgkin's lymphoma cells (SOX3-negative) . (3) Conduct competition assays using recombinant SOX3 protein to block specific binding while leaving cross-reactive signals unaffected. (4) Implement genetic models with SOX3 knockdown/knockout as definitive negative controls - studies comparing SOX3 immunostaining on Sox3-null cells from 14.5 dpc +/− embryos confirmed antibody specificity . (5) Use knockout/knockdown validation alongside Western blot analysis to confirm antibody specificity at the expected molecular weight. (6) For tissues expressing multiple SOX proteins, consider using a panel of antibodies against different SOX family members in adjacent sections to create an "expression map" that helps distinguish between family members with overlapping expression domains. (7) Consider alternative detection methods such as RNAscope in situ hybridization which offers higher specificity for transcript detection when protein-level specificity is challenging.

How can SOX3-FITC antibodies be integrated into studies of reproductive development and fertility?

SOX3-FITC antibodies can be powerfully applied to reproductive development research through several advanced approaches: (1) Implement co-immunostaining of SOX3 with steroidogenic enzymes in follicle development studies, particularly focusing on Cyp19a1a which is directly regulated by SOX3 and promotes 17β-estradiol synthesis that inhibits apoptosis in follicle development . (2) Combine TUNEL assays with SOX3 detection to visualize the relationship between SOX3 expression and apoptosis patterns in follicular cells - studies in sox3−/− zebrafish revealed obvious apoptosis signals in somatic cells (theca and granulosa cells) of stage III and IV follicles . (3) Establish SOX3 expression time courses in gonadal development across multiple vertebrate models, as SOX3 functions differ between species - it determines maleness in medaka fish and its disruption causes ZW female-to-male sex reversal in frogs . (4) Apply SOX3-FITC antibodies in chromatin immunoprecipitation studies to identify direct target genes beyond cyp19a1a that mediate SOX3's effects on reproduction. (5) Develop quantitative imaging protocols that correlate SOX3 expression levels with follicle development metrics, allowing statistical analysis of the relationship between SOX3 expression intensity and reproductive outcomes. (6) Implement cell sorting of SOX3-FITC labeled populations followed by transcriptomic analysis to comprehensively characterize SOX3-expressing cells in reproductive tissues.

What methodological approaches can be used to study the interaction between SOX3 and other transcription factors in gene regulation networks?

To investigate SOX3 interactions within transcriptional networks, researchers should consider these sophisticated methodological approaches: (1) Implement sequential chromatin immunoprecipitation (Re-ChIP) using SOX3 antibodies followed by immunoprecipitation with antibodies against putative partner transcription factors to identify co-occupied genomic loci. (2) Combine SOX3-FITC immunostaining with proximity ligation assays (PLA) to visualize and quantify in situ protein-protein interactions between SOX3 and candidate partners in the nuclear compartment. (3) Utilize reporter assays with systematic mutations of binding sites, as demonstrated in studies showing SOX3-26ala from mouse and human retains transactivation activity despite protein level reductions . (4) Develop CRISPR/Cas9-based approaches to tag endogenous SOX3 and partner proteins for live-cell imaging of transcription factor dynamics. (5) Apply quantitative mass spectrometry following SOX3 immunoprecipitation to identify the complete interactome in different developmental contexts. (6) For assessment of target gene regulation, combine SOX3 ChIP-seq with RNA-seq after SOX3 manipulation, as implemented in studies comparing sox3−/− and wild-type ovaries that revealed involvement in ovarian steroidogenesis and apoptosis pathways . (7) Implement ATACseq in SOX3+ versus SOX3- cell populations to identify chromatin accessibility changes mediated by SOX3, providing insights into its pioneer factor potential.

How can researchers effectively use SOX3-FITC antibodies in combination with genetic manipulation techniques to study SOX3 function in development?

Integrating SOX3-FITC antibodies with genetic manipulation techniques enables sophisticated functional analysis: (1) Establish chimeric embryo models combining wild-type and SOX3-mutant cells, then use SOX3-FITC antibodies to track cell-autonomous versus non-cell-autonomous effects, as demonstrated in studies of Sox3-26ala<->WT chimeric telencephalon . (2) Implement CRISPR/Cas9 or TALEN approaches targeting different domains of SOX3 (HMG domain versus transcription activation domain) to create domain-specific mutations, then use antibodies to assess resulting protein expression and localization patterns . (3) Develop conditional knockout models with temporal control to distinguish between developmental versus homeostatic SOX3 functions, using antibodies to confirm deletion efficiency in specific tissues. (4) Combine lineage tracing of SOX3-expressing cells (using Sox3-CreERT2 systems) with antibody detection to determine the developmental fate of cells that expressed SOX3 at specific developmental timepoints. (5) Implement rescue experiments with structure-function analysis by introducing variants of SOX3 into knockout backgrounds, then using antibodies to correlate protein expression levels with phenotypic rescue. (6) For in vitro models, apply SOX3-FITC antibodies to track SOX3 expression during directed differentiation of stem cells under varied conditions, as performed in studies differentiating ES cells in CDM as multi-cellular bodies for 5 days to detect rare SOX3 positive cells in Sox3-26ala mutants .