SATB2 Antibody，FITC conjugated

What is SATB2 and why is it important in research?

SATB2 (Special AT-rich Sequence-Binding Protein 2) is a nuclear matrix-associated protein that binds to DNA at nuclear matrix- or scaffold-associated regions. It recognizes the sugar-phosphate structure of double-stranded DNA rather than specific nucleotide sequences . SATB2 functions as a transcription factor that controls nuclear gene expression by binding to matrix attachment regions (MARs) of DNA and inducing local chromatin-loop remodeling . It acts as a docking site for several chromatin remodeling enzymes and recruits corepressors (HDACs) or coactivators (HATs) directly to promoters and enhancers . SATB2 plays critical roles in neurodevelopment, particularly in the initiation of upper-layer neurons (UL1) genetic programs and inactivation of deep-layer neurons (DL) and UL2 specific genes . Additionally, it influences osteoblast differentiation, palate formation, and has been implicated in cancer progression, notably in glioblastoma .

What are the advantages of using FITC-conjugated SATB2 antibodies?

FITC (Fluorescein Isothiocyanate) conjugation offers several methodological advantages for SATB2 detection. The direct conjugation eliminates the need for secondary antibodies, reducing experimental steps, background noise, and cross-reactivity concerns . FITC emits green fluorescence (excitation ~495 nm, emission ~519 nm), making it compatible with standard fluorescence microscopy setups and allowing for multiplexing with other fluorophores in different spectral ranges. The conjugation provides spatial resolution for visualizing SATB2's nuclear localization, enabling researchers to observe its distribution patterns in relation to chromatin organization. For time-sensitive experiments, FITC-conjugated antibodies offer immediate visualization without additional incubation periods required for secondary antibody binding.

How should researchers select appropriate SATB2 antibody clones and epitopes?

Selection of SATB2 antibody clones and epitopes should be guided by specific experimental requirements:

• Epitope consideration: Available SATB2 antibodies target different regions including AA 228-369, AA 451-485, AA 540-620, and C-terminal regions . Select epitopes based on:

  • Domain-specific functionality research needs

  • Potential post-translational modifications near the epitope

  • Sequence conservation across species (if conducting cross-species studies)

• Host and clonality factors:

  • Polyclonal antibodies (like rabbit polyclonal) offer broader epitope recognition but potential batch variation

  • Monoclonal antibodies (such as EPNCIR130A) provide consistent specificity but may have limited epitope access

• Validation documentation: Prioritize antibodies validated through multiple techniques including Western blot, immunohistochemistry, and ideally knockout validation

What are the typical applications for FITC-conjugated SATB2 antibodies?

FITC-conjugated SATB2 antibodies support multiple methodological approaches:

• Immunofluorescence microscopy: Enables visualization of subcellular localization patterns of SATB2 within the nucleus, particularly at matrix attachment regions

• Flow cytometry (FACS): Allows quantitative assessment of SATB2 expression levels across cell populations and can be combined with other cellular markers

• Immunohistochemistry: Particularly on paraffin-embedded (IHC-P) and frozen sections (IHC-fro) at recommended dilutions of 1:50-200

• Live cell imaging: For certain research applications requiring temporal resolution of SATB2 dynamics

• Chromatin immunoprecipitation followed by microscopy: To visualize specific DNA-protein interactions where SATB2 is involved

What are the optimal fixation and permeabilization methods for SATB2-FITC antibody staining?

Optimal fixation and permeabilization protocols for SATB2-FITC antibody staining should consider the nuclear localization of this protein and preservation of epitope accessibility:

• Fixation options:

  • 4% paraformaldehyde (10-15 minutes at room temperature) preserves cellular architecture while maintaining epitope accessibility

  • Methanol:acetone (1:1) fixation (10 minutes at -20°C) may enhance nuclear protein detection but can affect FITC fluorescence quality

• Permeabilization protocol:

  • For paraformaldehyde-fixed samples: 0.1-0.3% Triton X-100 (10 minutes) or 0.5% Saponin

  • Graduated ethanol series may be used for gentle permeabilization while preserving nuclear structural integrity

• Antigen retrieval considerations:

  • Heat-induced epitope retrieval using citrate buffer (pH 6.0) for 15-20 minutes

  • For formalin-fixed paraffin-embedded tissues, a combination of heat and enzymatic treatment may be necessary

• Buffer optimization:

  • PBS with 1-3% BSA as blocking and antibody diluent

  • Avoid detergents that might extract nuclear matrix components relevant to SATB2 binding

How can researchers design effective multiplex staining protocols with SATB2-FITC antibodies?

Designing multiplex staining with SATB2-FITC antibodies requires careful consideration of spectral compatibility, antibody cross-reactivity, and detection methods:

• Compatible fluorophore selection:

  • Pair FITC (green, emission ~519 nm) with:

    • DAPI or Hoechst for nuclear counterstaining (blue)

    • Alexa Fluor 594/647 or similar red/far-red fluorophores for additional markers

• Sequential staining protocol:

  • Start with the weakest signal antibody first

  • Apply SATB2-FITC at manufacturer-recommended dilutions (typically 1:50-200)

  • Include intervening wash steps to minimize cross-reactivity

• Cross-reactivity prevention:

  • Use antibodies raised in different host species

  • Implement additional blocking steps between antibody applications

  • Consider Fab fragments to block remaining active sites

• Technical considerations:

  • Account for FITC's susceptibility to photobleaching by minimizing exposure and using antifade mounting media

  • Document spectral bleed-through with single-stained controls for accurate analysis

What controls are essential when using SATB2-FITC antibodies?

Implementing appropriate controls ensures experimental rigor when using SATB2-FITC antibodies:

• Positive controls:

  • Cell lines/tissues with confirmed SATB2 expression (e.g., cortical neurons, osteoblasts)

  • Recombinant SATB2-expressing systems

• Negative controls:

  • Isotype controls with matching host species IgG-FITC conjugate

  • SATB2 knockout or knockdown samples

  • Primary antibody omission

• Specificity controls:

  • Peptide competition/blocking with the immunizing peptide

  • Parallel staining with alternative SATB2 antibody clones

  • Western blot validation to confirm specific band detection

• Technical controls:

  • Autofluorescence control (unstained sample)

  • Fluorescence minus one (FMO) controls for flow cytometry applications

  • Cross-adsorbed secondary antibodies for sequential staining protocols

How can SATB2-FITC antibodies be utilized to study chromatin organization?

SATB2-FITC antibodies offer powerful tools for investigating chromatin architecture due to SATB2's role in binding matrix attachment regions:

• Chromatin loop visualization methodology:

  • Combine SATB2-FITC staining with DNA fluorescence in situ hybridization (FISH) for specific genomic regions

  • Implement super-resolution microscopy techniques (STORM, PALM) to resolve fine chromatin structures

  • Use proximity ligation assays (PLA) to detect interactions between SATB2 and other chromatin remodeling factors

• Live-cell dynamics analysis:

  • Time-lapse imaging with SATB2-FITC antibody fragments to track chromatin reorganization

  • Fluorescence recovery after photobleaching (FRAP) to assess SATB2 binding kinetics to chromatin regions

• Chromatin accessibility studies:

  • Correlate SATB2-FITC localization with ATAC-seq or DNase-seq data

  • Combine with histone modification antibodies to assess chromatin states where SATB2 binds

• Three-dimensional nuclear architecture:

  • 3D reconstruction of SATB2 distribution relative to chromosome territories

  • Quantitative analysis of SATB2 clustering at nuclear matrix attachment sites

What methodologies combine SATB2-FITC detection with analysis of its regulatory targets?

Integrating SATB2-FITC detection with downstream target analysis provides insights into its regulatory functions:

• Combined ChIP-IF approaches:

  • Perform chromatin immunoprecipitation followed by immunofluorescence to visualize both SATB2 and its genomic binding sites

  • Quantify co-localization with transcriptional machinery components

• SATB2-target protein co-detection:

  • Multiplex staining of SATB2-FITC with antibodies against BCL11B, Ctip2, and FOXM1

  • Measure relative expression levels and spatial relationships

• Transcriptional activity correlation:

  • Combine SATB2-FITC immunostaining with RNA-FISH to visualize active transcription of target genes

  • Use nascent RNA labeling techniques (e.g., EU incorporation) to assess transcriptional activity

• Interaction network mapping:

  • Implement protein-fragment complementation assays with SATB2 and potential interaction partners

  • Apply proximity-dependent biotinylation (BioID) followed by fluorescent streptavidin detection alongside SATB2-FITC

How are SATB2-FITC antibodies applied in cancer research, particularly glioblastoma studies?

SATB2-FITC antibodies have emerged as valuable tools in cancer research, with specific applications in glioblastoma studies:

• Cancer stem cell identification:

  • SATB2 is preferentially expressed in glioma stem cells (GSCs), making SATB2-FITC antibodies useful for isolating and characterizing this subpopulation

  • Flow cytometry protocols using SATB2-FITC can separate stem-like populations from bulk tumor cells

• Mechanistic pathway analysis:

  • Visualize SATB2 co-localization with CBP (CREB-binding protein) to assess activation of the FOXM1 pathway

  • Track nuclear localization dynamics in response to treatment with epigenetic modifiers

• Prognostic assessment protocols:

  • Quantify SATB2 expression levels and nuclear distribution patterns in patient samples

  • Correlate with clinical outcomes and treatment response data

• Therapeutic targeting validation:

  • Monitor changes in SATB2 expression and localization following experimental treatments

  • Assess disruption of SATB2-mediated transcriptional programs upon intervention

What are common issues with SATB2-FITC antibody staining and how can researchers resolve them?

Researchers may encounter several challenges when working with SATB2-FITC antibodies, with corresponding resolution strategies:

• Weak or absent signal:

  • Increase antibody concentration (within 1:50-200 range)

  • Enhance epitope accessibility with optimized antigen retrieval methods

  • Extend incubation time (overnight at 4°C)

  • Check sample fixation protocol for epitope preservation

• High background fluorescence:

  • Implement additional blocking steps (5-10% normal serum from the antibody host species)

  • Include 0.1-0.3% Triton X-100 in wash buffers

  • Reduce primary antibody concentration if signal-to-noise ratio is poor

  • Use low-autofluorescence mounting media

• Non-specific binding:

  • Validate antibody specificity with Western blotting

  • Pre-adsorb antibody with non-specific proteins

  • Include competitors for non-specific binding sites (non-fat dry milk, BSA)

• Photobleaching:

  • Minimize exposure time during imaging

  • Use anti-fade mounting media containing radical scavengers

  • Consider acquiring images of regions of interest first before extended sample scanning

How can signal-to-noise ratio be optimized when using SATB2-FITC antibodies?

Optimizing signal-to-noise ratio for SATB2-FITC antibody applications involves multiple technical considerations:

• Sample preparation refinements:

  • Fresh preparation of fixatives to ensure optimal chemical reactivity

  • Careful temperature control during fixation to prevent epitope masking

  • Rigorous washing between steps (minimum 3×5 minutes in PBS with gentle agitation)

• Antibody titration protocol:

  • Perform systematic dilution series (e.g., 1:50, 1:100, 1:200)

  • Quantify signal-to-noise ratio at each concentration

  • Select concentration that maximizes specific signal while minimizing background

• Microscopy settings optimization:

  • Adjust detector gain and offset to maximize dynamic range

  • Implement deconvolution algorithms for improved resolution

  • Use confocal microscopy with appropriate pinhole settings to reduce out-of-focus light

• Fluorescence enhancement strategies:

  • Apply tyramide signal amplification for low-abundance targets

  • Consider photobleaching-resistant fluorophores for extended imaging

  • Implement spectral unmixing for accurate signal separation

How should researchers address cross-reactivity concerns with SATB2 antibodies?

Addressing cross-reactivity concerns requires systematic validation and optimization approaches:

• Cross-reactivity assessment methodology:

  • Validate on multiple cell/tissue types with known SATB2 expression profiles

  • Compare staining patterns across antibodies targeting different SATB2 epitopes

  • Perform Western blotting to confirm single band detection at expected molecular weight

• Antibody selection criteria:

  • Prioritize affinity-purified antibodies (>95% purity)

  • Consider antibodies validated against recombinant SATB2 protein fragments

  • Review cross-reactivity documentation across human, mouse, and rat samples

• Experimental design modifications:

  • Include blocking peptides corresponding to the immunizing antigen

  • Implement stringent washing conditions (higher salt concentration or mild detergents)

  • Consider monoclonal antibody alternatives for highly specific applications

How should quantitative data from SATB2-FITC immunofluorescence be analyzed?

Quantitative analysis of SATB2-FITC immunofluorescence requires rigorous methodological approaches:

• Nuclear localization quantification:

  • Measure nuclear:cytoplasmic signal ratio to confirm proper localization

  • Analyze subnuclear distribution patterns (peripheral vs. central)

  • Quantify co-localization with nuclear matrix markers using Pearson's or Mander's coefficients

• Expression level analysis:

  • Establish standardized image acquisition parameters across samples

  • Normalize SATB2-FITC signal intensity to nuclear area or DNA content

  • Apply appropriate background subtraction algorithms

• Statistical approaches:

  • Implement batch analysis to minimize subjective bias

  • Use appropriate statistical tests based on data distribution

  • Establish thresholds for positive/negative classification based on control samples

• Bioinformatic integration:

  • Correlate SATB2 localization data with genomic binding sites from ChIP-seq

  • Integrate with transcriptomic data to assess functional impact

  • Develop machine learning approaches for pattern recognition in SATB2 distribution

What standards should be applied when publishing research using SATB2-FITC antibodies?

Publication of research using SATB2-FITC antibodies should adhere to specific reporting standards:

• Materials documentation requirements:

  • Full antibody details: manufacturer, catalog number, clone identifier, lot number

  • Epitope information (e.g., AA 228-369, C-terminal)

  • Dilution factors and incubation conditions used (1:50-200)

• Validation evidence:

  • Images of positive and negative controls

  • Comparison with alternative detection methods when possible

  • Specificity confirmation through appropriate controls

• Imaging parameters documentation:

  • Complete microscope specifications and settings

  • Detector configuration and image acquisition parameters

  • Any post-processing algorithms applied with justification

• Quantification methodology:

  • Detailed description of analysis pipeline

  • Software tools used with version information

  • Blinding procedures for unbiased analysis