ARID4B Antibody

What is ARID4B and what cellular functions does it regulate?

ARID4B (AT-rich interactive domain 4B) is a 1312 amino acid protein that belongs to the ARID gene family. It contains one ARID domain and is also known as BRCAA1 or SAP180 . ARID4B functions as a transcriptional coactivator for androgen receptor (AR) and retinoblastoma (RB) protein, playing integral roles in their regulatory pathways . Studies in mouse models demonstrate that ARID4B, along with its homolog ARID4A, regulates Sertoli cell function, spermatogenesis, and the blood-testis barrier integrity . Recent research has also identified ARID4B's involvement in the PTEN-PI3K pathway in prostate cancer, where it directly binds to and regulates the promoters of PI3K subunit genes PIK3CA and PIK3R2 .

What applications can ARID4B antibody be used for in research?

ARID4B antibody (such as 24499-1-AP) can be utilized in multiple experimental applications:

The antibody has been validated in several published studies for applications including WB, IP, and knockdown/knockout verification experiments .

How should ARID4B antibody be stored and handled for optimal performance?

ARID4B antibody should be stored at -20°C, where it remains stable for one year after shipment . The antibody is typically supplied as a liquid in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . For smaller sizes (20μl), the solution may contain 0.1% BSA. Aliquoting is unnecessary for -20°C storage, but may be beneficial for preventing freeze-thaw cycles if frequent use is anticipated . When working with the antibody, proper laboratory safety procedures should be followed, particularly due to the presence of sodium azide.

What are the optimal antigen retrieval methods for ARID4B immunohistochemistry?

For immunohistochemical applications with ARID4B antibody, the suggested antigen retrieval method is with TE buffer at pH 9.0 . Alternatively, citrate buffer at pH 6.0 can be used for antigen retrieval, though results may vary depending on tissue type and fixation methods . When working with challenging tissue samples, optimization experiments comparing both retrieval methods is recommended. For human testis tissue and cancer tissues (pancreatic, breast, and cervical), the TE buffer method has demonstrated consistent positive staining results . The antigen retrieval step is critical for exposing epitopes that may be masked during fixation processes.

How can I validate ARID4B antibody specificity in my experimental system?

Validating ARID4B antibody specificity requires a multi-faceted approach:

• Knockdown/knockout controls: Utilize CRISPR-Cas9 gene editing to generate ARID4B knockout cells as negative controls, as demonstrated in PC3 cells for ChIP-qPCR validation .

• Expected molecular weight verification: ARID4B has a calculated molecular weight of 148 kDa (1312 amino acids), but is typically observed as bands at approximately 100 kDa and 200 kDa in Western blot applications .

• Cross-reactivity assessment: The antibody shows tested reactivity with human samples and cited reactivity with both human and mouse samples .

• Positive controls: Include known positive samples such as MCF-7 cells, which consistently show detection in multiple applications (WB, IP, IF/ICC) .

• Peptide competition assay: Pre-incubating the antibody with the immunizing peptide (ARID4B fusion protein Ag21462) should abolish specific signals in your experimental system .

What are the critical parameters for successful ChIP experiments using ARID4B antibody?

For chromatin immunoprecipitation (ChIP) experiments investigating ARID4B binding to target gene promoters:

• Optimal crosslinking conditions: Standard 1% formaldehyde for 10 minutes at room temperature is typically sufficient for nuclear proteins like ARID4B.

• Sonication parameters: Adjust sonication conditions to generate DNA fragments between 200-500 bp.

• Antibody amount: Use 2-5 μg of ARID4B antibody per ChIP reaction, as this range has demonstrated successful immunoprecipitation in published studies .

• Controls: Include IgG control immunoprecipitations and samples from ARID4B knockout cells for specificity validation.

• Target selection: Focus on transcription start sites (TSSs) of genes, as ChIP-Seq analysis has shown enrichment of ARID4B binding near TSSs of target genes .

• Validation: Perform ChIP-qPCR with primers targeting the promoter regions of known ARID4B targets like PIK3CA and PIK3R2, while using non-target genes like MYOD1 or GAPDH as negative controls .

How do ARID4A and ARID4B functionally interact in transcriptional regulation?

ARID4A and ARID4B are homologous members of the ARID gene family that physically interact with each other and function cooperatively in transcriptional regulation . Studies using knockout mouse models demonstrate that:

• Functional redundancy: ARID4A and ARID4B show partial functional redundancy, with combined deficiency (Arid4a−/−Arid4b+/−) producing more severe phenotypes than single gene knockout .

• Coactivator function: Both proteins function as transcriptional coactivators for androgen receptor (AR) and retinoblastoma protein (RB), enhancing their transcriptional activity .

• Synergistic effects: Cotransfection of both ARID4A and ARID4B with AR further enhances activation of target gene promoters (e.g., Cldn3) compared to either protein alone .

• Context-dependent roles: While ARID4B has been reported as a component of the SIN3A/HDAC1 repressor complex, it functions as a coactivator in certain contexts, particularly in AR-mediated gene regulation .

For investigating these interactions, co-immunoprecipitation followed by Western blotting has been successfully employed to demonstrate physical interactions between ARID4A, ARID4B, AR, and RB proteins .

What is the significance of ARID4B in the PI3K pathway and how can this be experimentally investigated?

ARID4B has been identified as a regulator of the PI3K pathway through direct transcriptional regulation of PI3K subunit genes:

• Transcriptional regulation: ARID4B directly binds to the promoters of PIK3CA and PIK3R2 genes, which encode the p110α catalytic and p85β regulatory subunits of PI3K, respectively .

• Functional consequence: Knockdown of ARID4B decreases expression of p110α and p85β in prostate cancer cell lines (PC3, DU145, LNCaP), affecting PI3K pathway activation .

• Regulatory network: ARID4B functions within a PTEN-ARID4B-PI3K regulatory axis, which may be particularly relevant in prostate cancer contexts .

To investigate this pathway experimentally:

• ChIP-Seq and RNA-Seq: Combine chromatin immunoprecipitation sequencing with transcriptome analysis to identify direct ARID4B targets and their expression changes upon ARID4B manipulation .

• Promoter activity assays: Utilize luciferase reporter assays with PIK3CA and PIK3R2 promoters to quantify ARID4B-mediated transcriptional regulation .

• Pathway activation analysis: Measure phosphorylation of downstream PI3K targets (AKT, S6K, 4E-BP1) following ARID4B knockdown or overexpression.

• Functional outputs: Assess cellular processes regulated by PI3K (proliferation, migration, survival) in response to ARID4B modulation.

How can apparent discrepancies in ARID4B molecular weight be resolved in Western blot experiments?

Researchers often observe discrepancies between the calculated and observed molecular weights of ARID4B in Western blot experiments:

• Expected vs. observed weight: ARID4B has a calculated molecular weight of 148 kDa based on its 1312 amino acid sequence, but is typically observed at approximately 100 kDa and 200 kDa in Western blots .

• Potential explanations:

  • Post-translational modifications: Phosphorylation, SUMOylation, or other modifications may alter protein migration.

  • Alternative splicing: Different isoforms may be expressed in different cell types.

  • Protein-protein interactions: Strong interactions resistant to denaturation may cause altered migration.

  • Proteolytic processing: Partial proteolysis may generate specific fragments.

To resolve these discrepancies:

• Sample preparation optimization: Test different lysis buffers and denaturation conditions.

• Use of multiple antibodies: Compare results with antibodies targeting different epitopes of ARID4B.

• Mass spectrometry validation: Perform immunoprecipitation followed by mass spectrometry to confirm the identity of observed bands.

• Knockout/knockdown controls: Include ARID4B-depleted samples to confirm band specificity.

• Isoform-specific PCR: Determine which ARID4B transcript variants are expressed in your experimental system.

What are common pitfalls in immunoprecipitation experiments with ARID4B antibody?

When performing immunoprecipitation (IP) with ARID4B antibody, researchers should consider these common pitfalls and solutions:

• Insufficient antibody amount: For optimal results, use 0.5-4.0 μg of ARID4B antibody for 1.0-3.0 mg of total protein lysate .

• Inefficient cell lysis: ARID4B is predominantly nuclear, requiring efficient nuclear extraction. Use nuclear extraction buffers containing 0.1-0.5% NP-40 or similar non-ionic detergents.

• Protein-protein interaction disruption: To preserve interactions between ARID4B and its binding partners (like ARID4A, AR, or RB), use mild lysis conditions and avoid harsh detergents.

• High background: Increase washing stringency gradually, using buffers with increasing salt concentrations (150 mM to 500 mM NaCl).

• Antibody cross-reactivity: Include appropriate controls such as IgG IP and lysates from ARID4B knockout cells.

• Protein degradation: Add protease inhibitors to all buffers and maintain samples at 4°C throughout the procedure.

MCF-7 cells have been validated as a positive control for ARID4B immunoprecipitation experiments .

How should I interpret immunohistochemical staining patterns for ARID4B in different tissues?

Interpretation of ARID4B immunohistochemical staining requires careful consideration of several factors:

• Subcellular localization: ARID4B is predominantly a nuclear protein, so nuclear staining should be expected in positive cells. Cytoplasmic staining may represent non-specific binding or alternative functions.

• Tissue-specific expression: ARID4B shows variable expression across tissues. It is highly expressed in testicular Sertoli cells and has been detected in various cancer tissues including pancreatic, breast, and cervical cancers .

• Cell type heterogeneity: Within tissues, ARID4B expression may vary by cell type. For example, in testes, ARID4B is mainly expressed in Sertoli cells rather than germ cells .

• Staining intensity variation: Consider both the percentage of positive cells and the intensity of staining when evaluating IHC results.

• Comparative analysis: Always include positive controls (such as testis tissue for Sertoli cells) and negative controls (antibody diluent only) in each staining run.

For optimal results, use the recommended dilution range of 1:500-1:2000 for IHC applications and perform antigen retrieval with TE buffer at pH 9.0, or alternatively with citrate buffer at pH 6.0 .

What experimental approaches can resolve contradictory results between different ARID4B antibody applications?

When facing contradictory results between different applications (e.g., positive WB but negative IHC), consider these experimental approaches:

• Epitope accessibility assessment: Different applications expose different epitopes. The ARID4B antibody (24499-1-AP) is raised against a fusion protein (Ag21462) , which may have differential accessibility in various applications.

• Fixation and processing effects: For IHC/IF applications, test different fixation methods and antigen retrieval protocols to optimize epitope exposure.

• Antibody concentration optimization: Perform titration experiments for each application. The recommended dilutions vary significantly between applications (WB: 1:500-1:1000; IHC: 1:500-1:2000; IF/ICC: 1:200-1:800) .

• Alternative antibody validation: Use multiple antibodies targeting different epitopes of ARID4B to cross-validate results.

• Genetic validation: Implement CRISPR/Cas9 knockout or siRNA knockdown approaches to confirm antibody specificity across applications .

• Recombinant protein controls: Include recombinant ARID4B protein as a positive control in Western blotting to establish detection limits.

• Transcript-protein correlation: Compare protein detection with mRNA expression analysis (qRT-PCR or RNA-Seq) to identify potential discrepancies.

How can ARID4B antibody be utilized to investigate its role in genomic imprinting?

Mouse model studies have demonstrated that ARID4B plays a role in genomic imprinting through regulation of epigenetic modifications . To investigate this function:

• Chromatin landscape analysis: Combine ARID4B ChIP-Seq with histone modification ChIP-Seq (H3K4me3, H3K9me3, H3K27me3) to map ARID4B association with imprinted loci.

• DNA methylation correlation: Perform ARID4B ChIP followed by bisulfite sequencing to determine if ARID4B binding correlates with DNA methylation patterns at imprinted regions.

• Protein complex identification: Use ARID4B antibody for immunoprecipitation followed by mass spectrometry to identify ARID4B-associated epigenetic modifiers at imprinted loci.

• Allele-specific binding analysis: Utilize allele-specific ChIP-Seq approaches to determine if ARID4B binds preferentially to maternal or paternal alleles of imprinted genes.

• Developmental dynamics: Apply ARID4B immunofluorescence in conjunction with imprinted gene FISH to track temporal changes in ARID4B association with imprinted loci during development.

These approaches can help elucidate the mechanistic role of ARID4B in establishing or maintaining genomic imprinting patterns.

What is the potential for ARID4B as a therapeutic target in cancer, and how can this be investigated?

Given ARID4B's role in regulating the PI3K pathway in prostate cancer and its expression in various cancer tissues , it presents a potential therapeutic target:

• Expression correlation with outcome: Analyze ARID4B protein expression using the validated antibody in tissue microarrays to correlate expression with patient outcomes across cancer types.

• Functional dependency screening: Use CRISPR-Cas9 screens in cancer cell panels to identify cancer types with specific dependence on ARID4B function.

• Mechanism of action studies: Utilize the antibody in ChIP-Seq and proteomic studies to map ARID4B regulatory networks in cancer contexts compared to normal tissues.

• Drug combination strategies: Investigate whether ARID4B inhibition (via genetic means) sensitizes cancer cells to existing PI3K pathway inhibitors or AR antagonists.

• Protein-protein interaction targeting: Use the antibody to validate disruption of critical ARID4B interactions (with ARID4A, AR, or RB) by small molecules or peptides.

• Biomarker development: Evaluate whether ARID4B expression or post-translational modifications correlate with response to specific cancer therapies.

These approaches may inform development of ARID4B-targeted therapeutic strategies or identify contexts where ARID4B status predicts treatment response.