SATB2 Recombinant Monoclonal Antibody

What is SATB2 and what are its primary biological functions?

SATB2 is a DNA binding protein that specifically recognizes nuclear matrix attachment regions (MARs). It functions as a transcription factor controlling nuclear gene expression by binding to MARs and inducing local chromatin-loop remodeling. SATB2 serves as a docking site for chromatin remodeling enzymes and recruits corepressors (HDACs) or coactivators (HATs) to promoters and enhancers . In the developing cerebral cortex, SATB2 is required for initiating upper-layer neurons (UL1) genetic programming while inactivating deep-layer neurons and UL2 specific genes. It also plays crucial roles in palate formation and acts as a regulatory determinant in skeletal development and osteoblast differentiation .

What are the key applications of SATB2 antibodies in research?

SATB2 antibodies have multiple research applications including: (1) Identification and characterization of colorectal carcinomas, where SATB2 expression correlates with good prognosis ; (2) Differentiation of neuroendocrine neoplasms of colon and rectum from other GI tract, pancreatic, and lung neuroendocrine neoplasms ; (3) Identification of tumors with osteoblastic differentiation ; (4) Study of neural development and cortical formation; (5) Investigation of SATB2-associated syndrome (SAS) ; and (6) Research into transcriptional regulation and chromatin architecture. These antibodies are employed in techniques including Western blotting, immunohistochemistry, immunocytochemistry/immunofluorescence, flow cytometry, and immunoprecipitation .

How can I validate the specificity of SATB2 antibodies in my experimental system?

Validation of SATB2 antibodies should include multiple approaches: (1) Use SATB2 knockout cell lines as negative controls, which should show absence of signal at the expected molecular weight (approximately 83 kDa) ; (2) Perform parallel experiments with secondary antibody-only controls to identify potential non-specific background staining ; (3) Include positive control tissues known to express SATB2, such as cerebral cortex or colorectal tissue samples ; (4) Verify band size in Western blot (expected at 83 kDa); and (5) Compare results across multiple antibody clones if available. For immunohistochemistry applications, optimize antigen retrieval conditions (e.g., Tris/EDTA buffer pH 9 with heat-mediated retrieval) to ensure specific nuclear staining patterns .

How can SATB2 antibodies be used to investigate the molecular mechanisms of SATB2-associated syndrome (SAS)?

For investigating SAS molecular mechanisms, researchers should: (1) Generate iPSC lines from SAS patients and differentiate them into relevant cell types (neurons, osteoblasts) to study cellular phenotypes; (2) Use SATB2 antibodies to assess protein expression levels, localization, and interactions in patient-derived cells versus controls; (3) Perform ChIP-seq with SATB2 antibodies to identify genomic binding sites and potential dysregulation in SAS; (4) Analyze the impact of different SATB2 mutations on protein function through immunoprecipitation studies to identify altered protein-protein interactions; and (5) Employ SATB2 antibodies in combination with other neural markers to investigate postnatal functions of SATB2 in memory and cognition, which might be affected in SAS patients . These approaches can help determine whether SAS results from simple haploinsufficiency or if mutated SATB2 alleles exert dominant-negative effects .

What are the best methodological approaches for studying SATB2 in the context of chromatin architecture and gene regulation?

When investigating SATB2's role in chromatin architecture: (1) Combine ChIP-seq using validated SATB2 antibodies with Hi-C or other chromosome conformation capture techniques to map SATB2-mediated chromatin loops; (2) Use sequential ChIP (Re-ChIP) to identify genomic regions where SATB2 co-localizes with other chromatin modifiers like HDACs or HATs; (3) Perform ATAC-seq in parallel with SATB2 ChIP-seq to correlate SATB2 binding with chromatin accessibility changes; (4) Use CUT&RUN or CUT&Tag as alternatives to traditional ChIP for higher resolution mapping of SATB2 binding sites; and (5) Implement CRISPR-mediated tagging of endogenous SATB2 followed by ChIP to avoid potential artifacts from antibody cross-reactivity. When analyzing data, focus on integrated multi-omics approaches that combine SATB2 binding patterns with gene expression changes, chromatin accessibility, and three-dimensional genome organization .

How can I determine if SATB2 mutations result in haploinsufficiency or dominant-negative effects?

To distinguish between haploinsufficiency and dominant-negative effects: (1) Generate isogenic cell lines with heterozygous SATB2 knockout versus knock-in of specific SAS mutations; (2) Compare protein expression levels of both mutant and wild-type alleles using allele-specific antibodies or epitope tagging; (3) Perform immunoprecipitation with SATB2 antibodies followed by mass spectrometry to identify altered protein interaction partners in mutant versus wild-type conditions; (4) Use ChIP-seq to compare genomic binding profiles between wild-type SATB2, haploinsufficient models, and dominant-negative models; and (5) Assess the ability of wild-type SATB2 overexpression to rescue cellular phenotypes in patient-derived cells . If phenotypes are primarily caused by reduced SATB2 levels (haploinsufficiency), increasing wild-type expression should ameliorate defects, whereas dominant-negative effects may persist despite wild-type overexpression .

What are the optimal conditions for using SATB2 antibodies in immunohistochemistry of different tissue types?

For optimal immunohistochemistry results with SATB2 antibodies: (1) For formalin-fixed paraffin-embedded tissues, use heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 at 100°C for 56 minutes ; (2) Dilute primary SATB2 antibodies to appropriate concentrations (1:150 for cerebral cortex, 2 μg/ml for colon tissue) ; (3) Incubate antibodies for optimal times (16 minutes at 37°C works well for automated platforms like Ventana DISCOVERY ULTRA) ; (4) Use appropriate detection systems such as OptiView DAB IHC Detection Kit for consistent results; (5) Always include positive controls (cerebral cortex, colorectal tissue) and negative controls (secondary antibody only); and (6) Counterstain with hematoxylin for nuclear contrast. Different tissue types may require optimization of these parameters—for instance, neural tissues might require longer antigen retrieval times than epithelial tissues. When analyzing results, focus on nuclear staining patterns, as SATB2 is primarily localized to the nucleus .

What troubleshooting approaches should I consider when SATB2 antibodies show unexpected results in Western blotting?

When troubleshooting Western blot issues with SATB2 antibodies: (1) Always use freshly prepared lysates to minimize protein degradation as recommended for SATB2 detection ; (2) Optimize blocking conditions—5% non-fat dry milk in TBST is generally effective for reducing background ; (3) If signal is weak, consider increasing antibody concentration (1:1000 to 1:10000 dilutions have been successfully used) ; (4) For multiple bands, verify specificity using knockout controls and consider whether you might be detecting different SATB2 isoforms or post-translationally modified forms; (5) If no signal is detected, verify SATB2 expression in your sample type (certain cell lines like Saos-2 and SW1353 are known to express SATB2) ; and (6) If experiencing high background, try reducing secondary antibody concentration or increasing washing steps. Also consider using specialized Western blot protocols for nuclear proteins, as SATB2's nuclear localization might require modified extraction procedures .

How can I optimize flow cytometry protocols for intracellular SATB2 detection?

For successful flow cytometric detection of intracellular SATB2: (1) Use appropriate fixation and permeabilization reagents that maintain nuclear integrity while allowing antibody access—methanol/acetone fixation or commercial nuclear permeabilization kits are recommended; (2) Titrate antibody concentrations to determine optimal signal-to-noise ratio; (3) Include isotype controls matched to the SATB2 antibody to establish background staining levels; (4) Use known SATB2-positive and SATB2-negative cell populations as controls; (5) Consider co-staining with DNA dyes to confirm nuclear localization; and (6) When analyzing data, gate on intact, single cells and use bivariate plots to distinguish positive populations. For multiparameter experiments, perform fluorescence minus one (FMO) controls when combining SATB2 detection with other markers. Be aware that flow cytometry for nuclear targets like SATB2 typically shows higher coefficient of variation than membrane markers, requiring careful gating strategy development.

How reliable is SATB2 as a marker for colorectal origin in metastatic tumors of unknown primary site?

SATB2 is a valuable marker for identifying colorectal origin in metastatic contexts, with specific considerations: (1) SATB2, especially when combined with CK20, can identify almost all colorectal carcinomas ; (2) Upper gastrointestinal carcinomas and pancreatic ductal carcinomas typically show negative SATB2 staining, aiding in differential diagnosis ; (3) Ovarian carcinomas, lung adenocarcinomas, and adenocarcinomas from other origins rarely express SATB2, increasing its specificity for colorectal origin ; (4) When interpreting results, consider that while sensitivity is high, no single marker is 100% specific, necessitating use of SATB2 within a panel of markers; and (5) For optimal diagnostic accuracy, combine SATB2 immunohistochemistry with other colorectal markers (CDX2, CK20) and clinical data. When analyzing challenging cases, remember that SATB2 expression correlates with good prognosis in colorectal cancer, so negativity in poorly differentiated tumors doesn't necessarily exclude colorectal origin .

What is the current understanding of SATB2's role in central nervous system development and related disorders?

SATB2's functions in CNS development include: (1) Acting as a transcription factor controlling the development of upper-layer neurons in the cerebral cortex, specifically initiating UL1-specific genetic programs while repressing deep-layer neuron programs ; (2) Regulating corticocortical connections by repressing Ctip2 (BCL11B) expression ; (3) The SATB2 locus has been associated with schizophrenia risk and educational attainment through genome-wide association studies ; (4) SATB2-associated syndrome (SAS) presents with intellectual disability, absent or severely delayed speech, and behavioral issues that may worsen near puberty ; (5) Postnatal SATB2 functions appear to involve memory and cognition, distinct from its embryonic developmental roles . When studying SATB2 in neurodevelopmental contexts, researchers should consider both early developmental roles and postnatal functions, as interventions targeting postnatal functions may have greater therapeutic potential in conditions like SAS .

How can SATB2 antibodies be employed in investigating potential therapeutic approaches for SATB2-associated syndrome?

To investigate therapeutic approaches for SAS using SATB2 antibodies: (1) Use antibodies to screen for compounds that upregulate expression from the functional SATB2 allele in patient-derived cells; (2) Employ SATB2 immunostaining to evaluate the effectiveness of gene therapy approaches in restoring protein expression; (3) Investigate whether the structurally similar SATB1 protein could compensate for SATB2 function by comparing expression patterns and binding targets ; (4) For patients with nonsense mutations, use SATB2 antibodies to evaluate readthrough technologies that might produce full-length protein; (5) Assess antisense oligonucleotide approaches that could suppress mutant allele expression while preserving wild-type expression ; and (6) For postnatal therapy development, use SATB2 antibodies to determine exactly which cell types express SATB2 after birth and which isoforms predominate. These approaches should prioritize understanding which aspects of SAS might be treatable after birth, focusing on speech deficits, sleep disturbances, and seizures as key therapeutic targets .

How should researchers interpret SATB2 expression patterns across different tumor types?

When interpreting SATB2 expression across tumor types: (1) Strong nuclear SATB2 staining in colorectal carcinomas correlates with good prognosis and is present in most cases ; (2) In laryngeal squamous cell carcinoma, SATB2 functions as a tumor suppressor, with loss of expression correlating with high tumor grade and recurrence ; (3) SATB2 positivity in tumors with osteoblastic differentiation makes it a useful marker for bone-forming neoplasms ; (4) Upper GI carcinomas and pancreatic ductal carcinomas typically show negative staining, with rare SATB2 positivity in ovarian carcinomas, lung adenocarcinomas, and other adenocarcinomas ; (5) When analyzing neuroendocrine neoplasms, SATB2 positivity specifically identifies those of colorectal origin, as neuroendocrine tumors from other GI sites, pancreas, and lung are typically negative . Researchers should consider both the intensity and pattern of staining when interpreting results, as weak or focal positivity may have different diagnostic significance than strong, diffuse expression.

What are the key considerations for quantitative analysis of SATB2 expression in research applications?

For quantitative SATB2 expression analysis: (1) Establish consistent scoring systems for immunohistochemistry (e.g., H-score, Allred score, or percentage of positive cells) to enable comparison across studies; (2) For Western blot quantification, always normalize SATB2 signal to appropriate loading controls (GAPDH at 37 kDa has been validated) ; (3) When analyzing RNA expression data, be aware of potential differences between mRNA and protein levels due to post-transcriptional regulation; (4) Consider that different SATB2 isoforms may have distinct functions, necessitating isoform-specific analysis in some contexts; (5) For flow cytometry, report median fluorescence intensity rather than just percentage positive cells for more accurate quantification; and (6) When comparing expression across sample types or experimental conditions, ensure identical antibody concentrations, incubation times, and detection methods. Statistical analysis should account for the non-normal distribution typically observed with protein expression data.

How can researchers distinguish between different SATB2 mutations and their functional impacts?

To differentiate SATB2 mutations and their functional impacts: (1) Use validated SATB2 antibodies to assess protein expression, localization, and stability across different mutation types; (2) For truncating mutations, determine if nonsense-mediated decay occurs or if truncated proteins are produced by comparing RNA and protein levels; (3) For missense mutations, conduct immunoprecipitation studies to evaluate impacts on protein-protein interactions; (4) Employ ChIP-seq to compare DNA binding profiles between wild-type and mutant SATB2; (5) Analyze cellular phenotypes in isogenic cell lines expressing different SATB2 mutations to establish genotype-phenotype correlations ; and (6) Consider developing allele-specific antibodies for specific recurring mutations to directly study mutant protein behavior. When analyzing results, focus on distinguishing haploinsufficiency (reduced functional protein) from dominant-negative effects (mutant protein interfering with wild-type function) as these mechanisms have different therapeutic implications .