AIR1B Antibody

What is ARID1B and what cellular functions does it regulate?

ARID1B (AT-rich interactive domain 1B) is a subunit of the BRG1/BRM-associated factor (BAF) complex, also known as the mammalian switch/sucrose non-fermenting (SWI/SNF) complex. This chromatin remodeler plays essential roles in regulating organ development, tissue homeostasis, disease progression, and cancer biology . At the molecular level, ARID1B functions as an epigenetic modifier that maintains mesenchymal stem cell (MSC) homeostasis by regulating chromatin accessibility to control gene expression . ARID1B haploinsufficiency in humans causes Coffin-Siris syndrome, which is characterized by developmental delay, facial dysmorphism, and intellectual disability . While its role has been extensively studied in neuronal development, recent research has uncovered its critical function in stem cell regulation and maintenance .

What are the recommended methodologies for using ARID1B antibodies in immunoblotting experiments?

For optimal results in immunoblotting experiments with ARID1B antibodies:

• Sample preparation: Extract total protein from cells or tissues using RIPA buffer supplemented with protease inhibitors.

• Protein quantification: Use Bradford or BCA assay to ensure equal loading (25-50 μg protein per lane).

• Gel selection: Due to ARID1B's large size (236 kDa), use 6-8% SDS-PAGE gels or gradient gels.

• Transfer conditions: Employ wet transfer at low voltage (30V) overnight at 4°C to ensure complete transfer of high molecular weight proteins.

• Blocking: Use 5% non-fat milk or BSA in TBST for 1 hour at room temperature.

• Primary antibody: Dilute ARID1B antibody (e.g., 82979-4-PBS) at 1:1000 in blocking buffer and incubate overnight at 4°C .

• Detection: Use secondary antibodies compatible with your rabbit-derived ARID1B primary antibody .

• Controls: Include both positive controls (cell lines known to express ARID1B) and negative controls (ARID1B knockout cells or ARID1B-depleted samples).

How should researchers design ChIP experiments using ARID1B antibodies?

For chromatin immunoprecipitation (ChIP) experiments with ARID1B antibodies:

• Crosslinking: Fix cells with 1% formaldehyde for 10 minutes at room temperature.

• Chromatin preparation: Sonicate chromatin to fragments of 200-500 bp.

• Antibody selection: Use ChIP-validated ARID1B antibodies; recombinant monoclonal antibodies like 82979-4-PBS provide consistent results across experiments .

• IP conditions: Use 4-5 μg of ARID1B antibody per 25-30 μg of chromatin.

• Controls: Include IgG control, input samples, and positive control regions (known ARID1B binding sites).

• Washing: Perform stringent washes to reduce background.

• Analysis: Use qPCR for targeted analysis or next-generation sequencing for genome-wide profiling.

• Validation: Confirm ARID1B binding sites by testing multiple regions and using alternative approaches like CUT&RUN.

The key to successful ChIP experiments is optimizing antibody concentration and washing conditions for your specific cell type, as ARID1B occupancy may vary across different cellular contexts.

How can researchers effectively study ARID1B's role in stem cell maintenance using immunofluorescence techniques?

To investigate ARID1B's role in stem cell maintenance through immunofluorescence:

• Sample preparation:

  • For cultured MSCs: Fix with 4% paraformaldehyde for 15 minutes at room temperature.

  • For tissue sections: Use 10 μm cryosections or paraffin sections with appropriate antigen retrieval.

• Permeabilization and blocking:

  • Permeabilize with 0.2% Triton X-100 for 10 minutes.

  • Block with 5% normal serum from the species of your secondary antibody.

• Co-staining strategy:

  • Primary antibodies: Use rabbit anti-ARID1B antibody (1:100-1:500 dilution) along with markers for:

    • Stem cell quiescence: p27, p57, or FOXO3

    • MSC markers: CD73, CD90, CD105

    • Proliferation markers: Ki67 or EdU labeling

• Imaging considerations:

  • Use confocal microscopy to determine nuclear localization of ARID1B in relation to chromatin.

  • Perform Z-stack imaging to fully capture nuclear distribution.

• Quantification methods:

  • Measure ARID1B nuclear intensity.

  • Quantify co-localization with quiescence/proliferation markers.

  • Calculate percentage of cells expressing both ARID1B and stem cell markers.

This approach can reveal how ARID1B maintains mesenchymal stem cell quiescence, as recent research has shown that loss of ARID1B disturbs MSC quiescence and leads to their proliferation due to ectopic activation of non-canonical Activin signaling via p-ERK .

What is the optimal experimental design for investigating ARID1B's synthetic lethality relationship with ARID1A mutations?

To effectively investigate the synthetic lethality between ARID1B and ARID1A mutations:

• Cell line selection:

  • Use matched pairs of cell lines:

    • ARID1A-mutant cancer cell lines (e.g., OVISE, TOV21G)

    • ARID1A-wildtype cell lines (e.g., ES-2, 293T)

    • Generate isogenic cell lines using CRISPR/Cas9 to create ARID1A knockout in wildtype cells

• ARID1B depletion strategies:

  • Use at least 3 different shRNAs targeting different regions of ARID1B to control for off-target effects

  • Employ inducible shRNA or degron systems for temporal control

  • Consider partial knockdown to mimic therapeutic interventions

• Phenotypic assays:

  • Proliferation: Real-time cell analysis, MTT/WST-1 assays, or cell counting over 7-10 days

  • Colony formation: Assess both number and size of colonies after 14-21 days

  • Cell death: Annexin V/PI staining and flow cytometry analysis

  • Cell cycle analysis: PI staining and flow cytometry

• Molecular assessment:

  • BAF complex integrity: Co-immunoprecipitation of SWI/SNF complex components

  • Chromatin accessibility: ATAC-seq before and after ARID1B depletion

  • Transcriptional output: RNA-seq to identify differentially expressed genes

• In vivo validation:

  • Xenograft models with inducible ARID1B knockdown

  • Patient-derived xenografts from ARID1A-mutant tumors

This experimental design has successfully demonstrated that ARID1B is specifically required for the growth of ARID1A-mutant cancer cell lines, with ARID1B depletion impairing proliferation and colony formation in ARID1A-mutant cells but not in wildtype cells .

How can researchers integrate scRNA-seq and scATAC-seq with ARID1B antibody-based techniques to understand chromatin accessibility changes?

To integrate single-cell technologies with ARID1B antibody-based techniques:

• Experimental workflow:

  • Split samples for parallel processing:

    • Single-cell RNA-seq (scRNA-seq)

    • Single-cell ATAC-seq (scATAC-seq)

    • CUT&Tag or CUT&RUN with ARID1B antibodies

    • Immunofluorescence with ARID1B antibodies for cell sorting

• Cell preparation and sorting:

  • Use FACS to isolate specific cell populations based on surface markers

  • Consider index sorting to link sorted cells with their ARID1B expression levels

  • For mesenchymal stem cell studies, use GLI1+ cell sorting as described in recent research

• Single-cell multi-omics integration:

  • Perform scRNA-seq and scATAC-seq using compatible platforms

  • Use computational methods to integrate data (e.g., Seurat, Signac, ArchR)

  • Identify cell clusters with differential ARID1B expression/activity

• ARID1B-specific analyses:

  • Map ARID1B binding sites relative to open chromatin regions

  • Correlate ARID1B occupancy with gene expression changes

  • Identify transcription factor motifs enriched at ARID1B binding sites

  • Compare ARID1B-bound regions between different cell states

• Validation experiments:

  • Perform CUT&Tag with ARID1B antibodies on sorted cell populations

  • Use CRISPR/Cas9 to modify specific ARID1B binding sites identified

  • Perform ChIP-qPCR on target genes identified from single-cell analyses

This integrated approach was successfully applied to dissect the regulatory functions of ARID1B within mesenchymal stem cells using the mouse incisor model, revealing that ARID1B suppresses Bcl11b expression via specific binding to its third intron, unveiling direct inter-regulatory interactions among BAF subunits in MSCs .

How can researchers effectively use ARID1B antibodies in cancer immunohistochemistry studies?

For optimal immunohistochemistry (IHC) studies of ARID1B in cancer tissues:

• Tissue preparation:

  • Formalin-fixed paraffin-embedded (FFPE) sections: 4-5 μm thickness

  • Antigen retrieval: Citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) at 95-98°C for 20 minutes

• Antibody optimization:

  • Perform titration experiments (1:50 to 1:500) with recombinant monoclonal antibodies

  • Include positive control tissues (e.g., colon, neuronal tissues)

  • Use ARID1B-knockout tissues as negative controls

• Detection system:

  • Use polymer-based detection systems for enhanced sensitivity

  • Consider tyramide signal amplification for low-abundance detection

• Scoring methodology:

  • Assess both intensity (0-3) and percentage of positive cells

  • Calculate H-score (0-300) = Σ(intensity × percentage)

  • Document subcellular localization (nuclear vs. cytoplasmic)

• Multiplex IHC protocols:

  • For tumor microenvironment studies, combine ARID1B staining with:

    • Immune cell markers (CD4, CD8, CD68)

    • Cancer stem cell markers

    • Proliferation markers (Ki67)

  • Use multispectral imaging systems for analysis

• Correlation with genomic data:

  • Analyze ARID1B protein expression in relation to ARID1A mutation status

  • Compare expression patterns between MSI-high and MSS colorectal cancers

Researchers have used these approaches to demonstrate that aberrantly hypermethylated ARID1B is a novel biomarker in colorectal cancer, with strong evidence showing correlation between ARID1B expression and immune cell infiltration in the tumor microenvironment .

What experimental approaches can resolve conflicting data between ARID1B mRNA expression and protein detection in cancer samples?

To resolve discrepancies between ARID1B mRNA and protein levels in cancer samples:

• Comprehensive expression analysis:

  Method	Target	Purpose	Controls
  RT-qPCR	Specific ARID1B exons	Detect alternative splicing	Include primers spanning multiple exons
  Western blot	N and C-terminal epitopes	Detect protein truncation	Use antibodies recognizing different regions
  RNA-seq	Full transcript	Identify fusion transcripts	Include polyA and total RNA protocols
  Proteomics	ARID1B peptides	Quantify protein abundance	Include internal standards

• Post-transcriptional regulation analysis:

  • miRNA profiling: Identify miRNAs targeting ARID1B

  • RNA stability assays: Measure ARID1B mRNA half-life in different samples

  • Polysome profiling: Assess translational efficiency

• Post-translational regulation:

  • Ubiquitination studies: Immunoprecipitate ARID1B and probe for ubiquitin

  • Phosphorylation profiling: Use phospho-specific antibodies or mass spectrometry

  • Protein stability assays: Cycloheximide chase experiments

• Epigenetic regulation:

  • DNA methylation analysis of ARID1B promoter using bisulfite sequencing

  • ChIP-seq for histone modifications at the ARID1B locus

  • Correlation between ARID1B methylation and expression levels

• Integrated multi-omics approach:

  • Compare DNA methylation, mRNA expression, and protein levels in matched samples

  • Analyze TCGA data for correlations between ARID1B mRNA levels, methylation status, and patient survival

  • Identify samples with discordant mRNA/protein expression for deeper analysis

What are the optimal conditions for co-immunoprecipitation experiments to study ARID1B interactions with SWI/SNF complex components?

For successful co-immunoprecipitation (Co-IP) of ARID1B with SWI/SNF complex components:

• Lysis buffer optimization:

  • For intact complex isolation: Use gentle buffers containing 0.1-0.3% NP-40 or Digitonin

  • Buffer composition: 20 mM HEPES pH 7.9, 1.5 mM MgCl₂, 0.2 mM EDTA, 10% glycerol, 150 mM KCl

• Nuclear extraction protocol:

  • Perform separate cytoplasmic and nuclear extractions

  • Use nuclease treatment (Benzonase or DNase I) to release chromatin-bound complexes

• Antibody selection and validation:

  • Use recombinant monoclonal antibodies for reproducibility

  • Pre-clear lysates with protein A/G beads to reduce background

  • Include IgG control immunoprecipitations

• IP conditions:

  • Protein amount: 500-1000 μg nuclear extract per IP

  • Antibody amount: 4-5 μg per IP

  • Incubation: Overnight at 4°C with gentle rotation

• Detection strategy:

  • Probe for core SWI/SNF components: SMARCA4 (BRG1), SMARCC1/2, SMARCB1

  • Include antibodies against both canonical and non-canonical complex members

  • Compare complex composition between wildtype and ARID1A-mutant samples

• Validation approaches:

  • Reciprocal IP: Immunoprecipitate with antibodies against other complex members

  • Size exclusion chromatography: Confirm complex integrity

  • Density gradient centrifugation: Separate intact complexes from free subunits

This methodology has revealed that depletion of ARID1B in ARID1A-mutant cells results in dissociation of the core catalytic ATPase subunit SMARCA4 (BRG1) and reduced incorporation of several other subunits, while protein levels of core subunits such as SMARCA4, SMARCC2, and SMARCB1 were also decreased .

How can researchers effectively design CRISPR/Cas9 experiments to study ARID1B function?

For CRISPR/Cas9-based studies of ARID1B function:

• Guide RNA design strategy:

  • Target early exons (exons 1-3) for complete knockout

  • Design multiple sgRNAs per target region (minimum 4-6)

  • Use validated design tools (e.g., CRISPOR, CHOPCHOP)

  • Check for off-target effects with prediction algorithms

• Experimental approaches based on research goals:

  Research Goal	CRISPR Strategy	Cell System	Readout
  Complete knockout	Paired sgRNAs causing deletion	Immortalized cell lines	Western blot, RT-qPCR
  Domain-specific function	sgRNAs targeting specific domains	Primary cells	Protein interaction, ChIP-seq
  Regulatory element analysis	sgRNAs targeting intronic regions	Stem cells	Gene expression, ATAC-seq
  In vivo function	Conditional Cas9 expression	Mouse models	Tissue-specific phenotypes

• Advanced CRISPR applications:

  • CRISPRa/CRISPRi: For modulating ARID1B expression without editing

  • Base editing: For introducing specific mutations associated with disorders

  • Prime editing: For precise mutations or small insertions

  • CRISPR screening: To identify synthetic lethal interactions with ARID1B

• Validation strategies:

  • Sequence verification of edits

  • Western blot for complete protein loss

  • RNA-seq to confirm absence of cryptic splicing around the cut site

  • Off-target analysis using GUIDE-seq or similar methods

• Rescue experiments:

  • Re-express ARID1B wildtype to confirm specificity

  • Express specific domains to determine functional requirements

  • Use orthogonal approaches (shRNA) to confirm phenotypes

These approaches have been used to validate that ARID1B is a specific vulnerability in ARID1A-mutant cancers, showing that upon ARID1B depletion, protein levels of core SWI/SNF subunits decrease while mRNA levels remain largely unaffected .

What are the best practices for quantitative analysis of ARID1B expression in relation to immune cell infiltration in cancer samples?

For quantitative analysis of ARID1B expression in relation to immune infiltration:

• Comprehensive immune profiling approaches:

  • Multiplex immunofluorescence with ARID1B and immune markers

  • Mass cytometry (CyTOF) with ARID1B antibodies

  • Single-cell RNA-seq of tumor and immune compartments

  • Spatial transcriptomics to preserve tissue architecture

• Digital pathology workflow:

  • Whole slide scanning of ARID1B IHC and serial sections with immune markers

  • Cell segmentation using nuclear counterstain

  • Quantification of staining intensity per cell

  • Spatial analysis of ARID1B+ cells relative to immune infiltrates

• Bioinformatic methods for correlation analysis:

  • TIMER analysis to estimate immune subset infiltration levels

  • Pearson correlation between ARID1B expression and immune cell markers

  • Deconvolution algorithms (CIBERSORT, xCell) for immune composition

  • Integration with TCGA data for larger-scale analysis

• Statistical considerations:

  • Account for tumor heterogeneity by analyzing multiple regions

  • Use appropriate multiple testing corrections

  • Employ multivariate analysis to control for confounding factors

  • Consider non-linear relationships between ARID1B and immune variables

• Functional validation:

  • Co-culture experiments with ARID1B-modulated cancer cells and immune cells

  • In vivo models with immune component analysis

  • Cytokine profiling in relation to ARID1B expression

This methodology has revealed significant correlations between ARID1B expression and immune cell infiltration in colorectal cancer, with strong evidence showing negative correlation with T cells (CD8+ and CD4+) and NK cells, and positive correlation with B cells, macrophages, neutrophils, tumor-associated fibroblasts, dendritic cells, and regulatory T cells .

How should researchers address non-specific binding issues with ARID1B antibodies in western blotting?

To address non-specific binding with ARID1B antibodies in western blotting:

• Antibody validation strategy:

  • Test multiple antibodies targeting different epitopes

  • Use recombinant monoclonal antibodies for improved specificity

  • Include proper controls: ARID1B knockout/knockdown samples

  • Confirm band identity with mass spectrometry

• Protocol optimization:

  • Blocking: Test alternative blocking agents (5% milk vs. 5% BSA)

  • Washing: Increase stringency with higher detergent concentration (0.1-0.3% Tween-20)

  • Antibody concentration: Perform titration experiments (1:500 to 1:5000)

  • Incubation time: Reduce to minimize background (1-2 hours at room temperature vs. overnight)

• Sample preparation improvements:

  • Nuclear extraction: Ensure proper fractionation to enrich for nuclear proteins

  • Protein denaturation: Optimize temperature and time (70°C for 10 min vs. 95°C for 5 min)

  • Loading controls: Use nuclear-specific controls (Lamin B1 or Histone H3)

• Technical considerations for high molecular weight proteins:

  • Use lower percentage gels (6-8%) for better resolution

  • Extend transfer time for large proteins (overnight at low voltage)

  • Add SDS (0.1%) to transfer buffer to improve large protein transfer

  • Consider using PVDF membrane instead of nitrocellulose

• Peptide competition assay:

  • Pre-incubate antibody with immunizing peptide

  • Specific bands should disappear while non-specific bands remain

Using these approaches helps ensure specific detection of ARID1B, which has a calculated molecular weight of 236 kDa , making it particularly challenging for western blot analysis.

What strategies can researchers employ when facing inconsistent ARID1B chromatin immunoprecipitation results?

To troubleshoot inconsistent ARID1B ChIP results:

• Crosslinking optimization:

  • Test different formaldehyde concentrations (0.5-2%)

  • Evaluate crosslinking times (5-20 minutes)

  • Consider dual crosslinking with DSG or EGS before formaldehyde for protein-protein interactions

• Chromatin fragmentation assessment:

  • Optimize sonication conditions for consistent fragment size (200-500 bp)

  • Verify fragment size distribution by agarose gel or Bioanalyzer

  • Test different sonication buffers (SDS concentrations: 0.1-1%)

• Antibody evaluation:

  • Compare multiple ARID1B antibodies recognizing different epitopes

  • Validate antibody specificity using knockout/knockdown controls

  • Test different antibody amounts (2-10 μg per ChIP)

  • Consider using recombinant monoclonal antibodies for consistency

• Protocol improvements:

  • Pre-clear chromatin to reduce background

  • Adjust washing stringency based on signal-to-noise ratio

  • Use magnetic beads instead of agarose for cleaner pull-downs

  • Block beads with BSA and yeast tRNA to reduce non-specific binding

• Cell type and condition considerations:

  • ARID1B may have cell type-specific binding patterns

  • Consider cell cycle synchronization (ARID1B binding may vary across cell cycle)

  • Assess effect of cell culture conditions on chromatin structure

• Data analysis approaches:

  • Use appropriate negative control regions (gene deserts)

  • Consider sequence bias in ChIP-seq data analysis

  • Integrate with ATAC-seq or DNase-seq data to focus on accessible regions

  • Compare with published ARID1B ChIP-seq datasets as reference

These strategies can help researchers obtain consistent results when studying ARID1B binding to specific genomic regions, such as the third intron of Bcl11b, where ARID1B has been shown to suppress Bcl11b expression .