ARI2 Antibody

What is ARID2 and what cellular functions does it perform?

ARID2 (AT-rich interactive domain 2) is a 197 kDa protein involved in chromatin remodeling complexes. It functions as a subunit of the PBAF (Polybromo-associated BAF) complex, which regulates gene expression through ATP-dependent chromatin remodeling. The protein contains 1835 amino acids and plays critical roles in transcriptional regulation by facilitating accessibility of transcription factors to DNA. When working with ARID2 antibodies, it's important to understand that the observed molecular weight on western blots typically ranges from 200-230 kDa, which is slightly higher than the calculated weight due to post-translational modifications .

What applications can ARID2 antibody be used for?

ARID2 antibody has been validated for several experimental applications:

For optimal results in IHC applications, antigen retrieval with TE buffer at pH 9.0 is recommended, though citrate buffer at pH 6.0 may also be used as an alternative .

How should ARID2 antibody be stored and handled?

ARID2 antibody should be stored at -20°C where it remains stable for one year after shipment. The antibody is typically supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3. Aliquoting is not necessary for -20°C storage. For smaller 20μl sizes that contain 0.1% BSA, avoid repeated freeze-thaw cycles to maintain antibody activity. When handling the antibody, wear appropriate PPE due to the presence of sodium azide, which is toxic .

What models or sample types have been validated with ARID2 antibody?

ARID2 antibody has been tested and validated with:

When extending to untested samples, preliminary validation experiments are strongly recommended to confirm reactivity and specificity .

How can epitope mapping techniques be applied to characterize ARID2 antibody binding?

Epitope mapping for ARID2 antibody can be approached similarly to methods used for other antibodies, such as those described for ACE2 autoantibodies. A comprehensive approach would involve:

• Creating a peptide library spanning the entire ARID2 protein (1835 amino acids)

• Synthesizing overlapping peptides (e.g., 15 amino acids with 11 amino acid overlaps)

• Immobilizing peptides on glass microarray surfaces using hydrophilic linker moieties

• Incubating diluted antibody samples (typically 1:200) with the peptide microarray

• Detecting binding with fluorescently labeled secondary antibodies

• Analyzing binding patterns to identify specific epitopes

This approach can reveal which domains of ARID2 are recognized by the antibody, which is particularly valuable for determining if functional domains are targeted and if the antibody might interfere with protein-protein interactions .

What strategies can overcome cross-reactivity issues with ARID2 antibody?

When encountering cross-reactivity with ARID2 antibody, consider implementing these advanced troubleshooting strategies:

• Perform pre-adsorption tests by incubating the antibody with purified recombinant ARID2 protein before application to samples

• Increase blocking stringency by using 5% BSA or specialized blocking reagents designed for polyclonal antibodies

• Optimize antibody concentration through careful titration experiments

• Include additional washing steps with higher salt concentrations (up to 500 mM NaCl) to reduce non-specific binding

• Validate results with multiple antibodies targeting different epitopes of ARID2

• Consider using knockout or knockdown controls to definitively establish specificity

Cross-reactivity assessment is particularly important when studying closely related ARID family proteins that share sequence homology with ARID2 .

How can machine learning approaches improve antibody-antigen prediction for ARID2 studies?

Machine learning models can significantly enhance ARID2 antibody applications through improved prediction of binding characteristics:

• Library-on-library approaches can identify specific interacting pairs between ARID2 antibody variants and target epitopes

• Active learning algorithms can reduce experimental costs by starting with a small labeled subset of binding data and iteratively expanding only the most informative data points

• Out-of-distribution prediction can help estimate how ARID2 antibodies might interact with previously uncharacterized mutant proteins or related family members

• Simulation frameworks like Absolut! can be used to evaluate potential binding before committing to expensive experimental validation

Studies have shown that optimized active learning strategies can reduce the number of required antigen mutant variants by up to 35% and accelerate the learning process compared to random sampling approaches .

What are the considerations for using ARID2 antibody in multiplex immunofluorescence studies?

For multiplex immunofluorescence studies incorporating ARID2 antibody, researchers should address these critical factors:

• Antibody Clone Selection:

  • Verify the rabbit polyclonal ARID2 antibody (23406-1-AP) is compatible with your multiplexing approach

  • Consider potential cross-reactivity with other antibodies in your panel

• Signal Amplification:

  • For nuclear proteins like ARID2, tyramide signal amplification may be necessary due to lower abundance

  • Optimize signal-to-noise ratio through careful titration experiments

• Antigen Retrieval Compatibility:

  • Ensure all antibodies in the multiplex panel are compatible with the TE buffer pH 9.0 recommended for ARID2

  • If incompatibilities exist, sequential staining approaches may be required

• Spectral Overlap:

  • Design panels accounting for the nuclear localization of ARID2 to avoid spatial overlap with other nuclear markers

  • Perform single-color controls to establish proper unmixing parameters

• Validation Controls:

  • Include positive controls (Jurkat or K-562 cells) and negative controls (tissues with ARID2 knockdown) in multiplex panels

  • Perform parallel single-staining to confirm multiplex results .

How should Western blot protocols be optimized for ARID2 detection?

For optimal Western blot detection of ARID2, follow these methodological guidelines:

• Sample Preparation:

  • Use RIPA buffer supplemented with protease inhibitors for efficient extraction

  • Include phosphatase inhibitors if phosphorylation status is relevant

  • Heat samples at 95°C for 5 minutes in loading buffer containing SDS and DTT

• Gel Selection and Transfer:

  • Use 6-8% SDS-PAGE gels or gradient gels (4-15%) to resolve the 200-230 kDa ARID2 protein

  • Perform wet transfer at low voltage (30V) overnight at 4°C to ensure complete transfer of high molecular weight proteins

  • Use PVDF membrane (0.45 μm pore size) rather than nitrocellulose for better retention of large proteins

• Antibody Incubation:

  • Block membranes with 5% non-fat dry milk in TBST for 1 hour at room temperature

  • Dilute ARID2 antibody 1:500 in blocking buffer for overnight incubation at 4°C

  • Wash extensively (4 × 10 minutes) with TBST before secondary antibody incubation

• Detection Optimization:

  • Use enhanced chemiluminescence with extended exposure times (up to 5 minutes)

  • Consider gradient exposure times to capture the optimal signal intensity

  • For quantitative analysis, use fluorescently-labeled secondary antibodies and fluorescence imaging

This protocol has been validated using Jurkat and K-562 cell lysates, which serve as positive controls for ARID2 detection .

What controls are essential when using ARID2 antibody in research applications?

A robust experimental design using ARID2 antibody should incorporate these essential controls:

These controls help distinguish true positive signals from artifacts and allow proper interpretation of experimental results when using ARID2 antibody .

How can epitope accessibility be improved in fixed tissues for ARID2 immunohistochemistry?

Optimizing epitope accessibility for ARID2 IHC requires careful consideration of fixation and antigen retrieval methods:

• Fixation Optimization:

  • Limit fixation time with 10% neutral-buffered formalin to 24 hours to prevent excessive cross-linking

  • Consider alternative fixatives such as zinc-based fixatives that preserve epitopes while maintaining tissue morphology

  • For frozen sections, use acetone fixation for 10 minutes at -20°C to improve epitope preservation

• Antigen Retrieval Methods:

  • Heat-induced epitope retrieval (HIER) with TE buffer at pH 9.0 is recommended as the primary method

  • Alternative protocol: citrate buffer at pH 6.0 with pressure cooking for 15 minutes

  • Enzymatic retrieval using proteinase K (10 μg/ml for 10-15 minutes) may be tested if HIER is ineffective

• Signal Enhancement Strategies:

  • Implement tyramide signal amplification for low-abundance detection

  • Extend primary antibody incubation to overnight at 4°C using a 1:250 dilution

  • Add 0.1% Triton X-100 to antibody diluent to improve tissue penetration

• Background Reduction:

  • Include 10% normal goat serum in blocking buffer

  • Add 0.1% BSA to washing buffers to reduce non-specific binding

  • Consider avidin-biotin blocking steps if using biotin-based detection systems

These optimizations have proven effective for detecting ARID2 in mouse testis tissue and can be adapted for other tissue types .

How can researchers address weak or absent ARID2 signal in Western blots?

When encountering weak or absent ARID2 signals in Western blots, implement this systematic troubleshooting approach:

• Protein Extraction Assessment:

  • Verify complete lysis using stronger extraction buffers (e.g., urea-containing buffers)

  • Sonicate samples to improve chromatin-associated protein extraction

  • Confirm protein concentration using Bradford or BCA assays

• Transfer Efficiency Analysis:

  • Verify transfer of high molecular weight proteins using reversible stains (Ponceau S)

  • Reduce transfer time or voltage if protein is passing through the membrane

  • Consider semi-dry transfer systems optimized for high molecular weight proteins

• Antibody Optimization:

  • Increase primary antibody concentration (up to 1:250 dilution)

  • Extend primary antibody incubation to 48 hours at 4°C

  • Test alternative lots of the same antibody catalog number

• Sample-Specific Considerations:

  • Confirm ARID2 expression in your specific cell type or tissue through transcript analysis

  • Check literature for expected ARID2 expression levels in your experimental system

  • Consider that post-translational modifications may alter epitope accessibility

• Detection Enhancement:

  • Use high-sensitivity ECL substrates designed for low-abundance proteins

  • Increase exposure time during imaging (up to 30 minutes)

  • Consider using cooled CCD camera systems with integration capabilities

This approach has helped researchers successfully detect the 200-230 kDa ARID2 protein even in challenging sample types .

What are common sources of non-specific binding with ARID2 antibody and how can they be mitigated?

Non-specific binding is a common challenge when working with ARID2 antibody. Here are typical sources and mitigation strategies:

These strategies have been effective in reducing background and improving signal-to-noise ratio in ARID2 detection experiments .

How should researchers interpret apparent molecular weight discrepancies for ARID2?

The calculated molecular weight of ARID2 is 197 kDa, but the observed molecular weight on Western blots is typically 200-230 kDa. This discrepancy requires careful interpretation:

• Post-Translational Modifications:

  • Phosphorylation at multiple sites can significantly increase apparent molecular weight

  • Other modifications (glycosylation, SUMOylation) may contribute to shifts

  • Different cell types may exhibit varying modification patterns

• Technical Considerations:

  • Use molecular weight markers that extend beyond 200 kDa for accurate size determination

  • Run gels at lower voltage (80V) for extended time to improve resolution of high molecular weight proteins

  • Consider using gradient gels (4-15%) for better size separation in the high molecular weight range

• Validation Approaches:

  • Verify specificity through siRNA knockdown of ARID2, which should reduce the intensity of the true ARID2 band

  • Test antibody on samples from multiple tissues to establish a consistent molecular weight pattern

  • Compare results with alternative ARID2 antibodies targeting different epitopes

• Data Interpretation Guidelines:

  • Accept bands within the 200-230 kDa range as potentially legitimate ARID2 signal

  • Be cautious of bands below 190 kDa, which likely represent degradation products or non-specific binding

  • Document exact conditions when molecular weight varies to identify potential regulatory mechanisms

Understanding these molecular weight variations is crucial for proper data interpretation and can provide insights into the post-translational regulation of ARID2 .

How can machine learning approaches be applied to analyze ARID2 antibody binding data?

Machine learning techniques offer powerful tools for analyzing complex ARID2 antibody binding data:

• Binding Affinity Prediction:

  • Train models using library-on-library screening data to predict binding between antibody variants and target epitopes

  • Implement active learning strategies that iteratively identify the most informative experiments to perform

  • These approaches can reduce experimental costs by up to 35% compared to random sampling

• Epitope Mapping Analysis:

  • Apply clustering algorithms to identify patterns in peptide array binding data

  • Use feature importance analysis to identify key amino acid residues critical for antibody recognition

  • Implement transfer learning from similar antibody-antigen systems to improve prediction accuracy

• Cross-Reactivity Assessment:

  • Develop models that predict potential cross-reactivity with related proteins based on sequence similarity

  • Use out-of-distribution prediction techniques to estimate binding to novel mutant variants

  • Validate computational predictions with targeted experimental verification

• Experimental Design Optimization:

  • Implement Bayesian optimization to determine optimal antibody concentrations and incubation conditions

  • Use simulation frameworks to predict experimental outcomes before committing resources

  • Accelerate the learning process by approximately 28 steps compared to random experimental design

These machine learning approaches can significantly enhance research efficiency and improve the reliability of ARID2 antibody applications in complex experimental systems .

How can ARID2 antibody be used in chromatin immunoprecipitation (ChIP) studies?

ARID2 antibody can be effectively employed in ChIP studies to investigate chromatin remodeling complexes:

• ChIP Protocol Optimization:

  • Crosslink cells with 1% formaldehyde for 10 minutes at room temperature

  • Sonicate chromatin to fragments of 200-500 bp

  • Use 5 μg of ARID2 antibody per ChIP reaction

  • Incubate antibody-chromatin mixture overnight at 4°C with rotation

  • Include appropriate controls (IgG, input, positive control regions)

• Technical Considerations:

  • Pre-clear chromatin with protein A/G beads before antibody addition

  • Use sonication conditions optimized for nuclear proteins

  • Include detergents (0.1% SDS, 1% Triton X-100) in wash buffers

  • Verify fragment size distribution by agarose gel electrophoresis

• Downstream Applications:

  • Perform ChIP-seq to map genome-wide ARID2 binding sites

  • Use ChIP-qPCR to validate binding at specific genomic loci

  • Combine with RNA-seq to correlate binding with transcriptional outcomes

• Data Analysis Approach:

  • Use peak calling algorithms optimized for transcription factors

  • Perform motif analysis to identify DNA sequences associated with ARID2 binding

  • Integrate with public ChIP-seq datasets for other PBAF complex components

This methodology enables researchers to investigate ARID2's role in chromatin remodeling and transcriptional regulation at a genome-wide scale.

What considerations are important when using ARID2 antibody in protein-protein interaction studies?

For investigating ARID2 protein interactions, consider these methodological aspects:

• Co-Immunoprecipitation (Co-IP) Optimization:

  • Use gentle lysis buffers (e.g., 150 mM NaCl, 50 mM Tris pH 7.5, 0.5% NP-40) to preserve protein complexes

  • Pre-clear lysates thoroughly to reduce non-specific binding

  • Incubate 2-5 μg of ARID2 antibody with 500-1000 μg of protein lysate

  • Include appropriate controls (IgG pulldown, input, reverse Co-IP)

• Cross-Linking Strategies:

  • Consider reversible cross-linkers (DSP, DTBP) to stabilize transient interactions

  • Optimize cross-linker concentration and incubation time

  • Include controls to verify cross-linking efficiency

• Detection Methods:

  • Use reciprocal Co-IP to confirm interactions

  • Perform Western blot analysis with antibodies against known PBAF complex components

  • Consider mass spectrometry for unbiased identification of interaction partners

• Functional Validation:

  • Verify biological relevance through knockout/knockdown studies

  • Map interaction domains using truncation mutants

  • Assess interaction dynamics in response to cellular stimuli

These approaches enable comprehensive characterization of ARID2's role within the PBAF complex and identification of novel interaction partners.

What are the considerations for using ARID2 antibody in examining post-translational modifications?

To investigate post-translational modifications (PTMs) of ARID2:

• Sample Preparation:

  • Include phosphatase inhibitors (sodium orthovanadate, sodium fluoride) to preserve phosphorylation

  • Add deubiquitinase inhibitors (N-ethylmaleimide) for ubiquitination studies

  • Use HDAC inhibitors (sodium butyrate, trichostatin A) for acetylation analysis

• Enrichment Strategies:

  • Perform immunoprecipitation with ARID2 antibody followed by Western blot with PTM-specific antibodies

  • Consider phospho-enrichment using TiO₂ or IMAC prior to analysis

  • Use ubiquitin-specific pulldown (TUBE technology) for ubiquitination studies

• Detection Methods:

  • Employ PTM-specific antibodies (phospho, acetyl, ubiquitin, SUMO) in Western blots

  • Consider mass spectrometry for comprehensive PTM mapping

  • Use Phos-tag gels to separate phosphorylated from non-phosphorylated forms

• Functional Validation:

  • Correlate PTM status with ARID2 activity and localization

  • Generate site-specific mutants to determine PTM function

  • Investigate enzymes responsible for adding/removing specific PTMs

This methodological approach provides insights into how PTMs regulate ARID2 function within chromatin remodeling complexes.

What emerging technologies are enhancing ARID2 antibody applications in research?

Several cutting-edge technologies are expanding the utility of ARID2 antibody in advanced research applications:

• Proximity Labeling:

  • BioID or APEX2 fusions to study the ARID2 interactome in living cells

  • TurboID for rapid labeling of transient interaction partners

  • Split-BioID for detecting specific protein-protein interactions

• Super-Resolution Microscopy:

  • STORM/PALM techniques to visualize ARID2 localization at nanometer resolution

  • Expansion microscopy to physically enlarge samples for improved visualization

  • Lattice light-sheet microscopy for dynamic imaging of ARID2 in living cells

• Single-Cell Applications:

  • CUT&Tag for single-cell profiling of ARID2 chromatin binding

  • scRNA-seq combined with ARID2 perturbation for functional genomic analysis

  • Spatial transcriptomics to correlate ARID2 binding with gene expression in tissue context

• Artificial Intelligence:

  • Deep learning for improved image analysis in ARID2 immunofluorescence

  • Active learning strategies to optimize experimental design

  • Predictive modeling for antibody-antigen interactions

These technologies represent the frontier of ARID2 research and offer unprecedented insights into its biological functions and regulatory mechanisms .

How can researchers validate ARID2 antibody specificity across different experimental systems?

A comprehensive validation strategy for ARID2 antibody across experimental systems includes:

• Genetic Validation:

  • CRISPR/Cas9 knockout of ARID2 as negative control

  • siRNA knockdown to demonstrate signal reduction

  • Rescue experiments with exogenous ARID2 expression

• Multi-Antibody Approach:

  • Compare results using antibodies targeting different ARID2 epitopes

  • Correlate findings across multiple commercial antibodies

  • Use monoclonal and polyclonal antibodies to verify observations

• Cross-Species Validation:

  • Test reactivity in human, mouse, and other model organisms

  • Compare staining patterns across evolutionarily related species

  • Assess conservation of molecular weight and subcellular localization

• Multi-Technique Confirmation:

  • Correlate Western blot findings with immunohistochemistry results

  • Verify protein expression with transcript levels (RT-qPCR, RNA-seq)

  • Combine immunofluorescence with in situ hybridization

This rigorous validation approach ensures reliable and reproducible results across diverse experimental contexts and biological systems.