ARID4 Antibody

What are ARID4 proteins and why are they important research targets?

ARID4 belongs to the AT-rich interaction domain (ARID) family, which includes 15 members divided into seven subfamilies based on sequence identity. The ARID4 subfamily specifically contains two members: ARID4A and ARID4B. These proteins function as transcription regulators affecting cell growth, differentiation, and development . ARID4B, also known as BRCAA1 or SAP180, is a 1312 amino acid protein containing one ARID domain and localizes in both the nucleus and cytoplasm . ARID4 proteins interact with the retinoblastoma protein (pRB) and the mSIN3-histone deacetylase (mSIN3-HDAC) complex, playing crucial roles in cellular processes including transcriptional regulation and growth control . Their involvement in cancer-related signaling pathways makes them important targets for oncology research.

What is the structural and functional difference between ARID4A and ARID4B?

• ARID4A contains an additional LXCXE motif, which functions as an RB binding motif not present in ARID4B

• ARID4A represses E2F-dependent transcription by recruiting the mSIN3-HDAC complex to pRB family members, playing a central role in arresting cell growth

• ARID4B has been found to localize in both nucleus and cytoplasm, suggesting potentially distinct functions in different cellular compartments

• The genes are located on different chromosomes: ARID4A on 14q23.1 and ARID4B on 1q42.1-q43

These differences explain their distinct roles in cellular processes and why researchers might target one over the other depending on the biological question.

What expression patterns of ARID4B should researchers be aware of when designing experiments?

ARID4B exhibits tissue-specific expression patterns that researchers should consider when designing experiments. According to available data, ARID4B is:

• Highly expressed in testis and in breast, lung, colon, pancreatic and ovarian cancers

• Expressed at low levels in thymus, prostate and ovary

• Detected in various cell lines including MCF-7 (breast cancer), A549 (lung cancer), and K-562 (leukemia) cells

Understanding these expression patterns is crucial when selecting appropriate experimental models and interpreting results. For instance, using cell lines with known ARID4B expression levels as positive controls can help validate antibody specificity and experimental conditions.

What are the validated applications for ARID4B antibodies and their recommended protocols?

Based on extensive validation data, ARID4B antibodies have been successfully applied in multiple experimental techniques. The polyclonal antibody (24499-1-AP) has been validated for:

For the monoclonal antibody (67384-1-PBS), validated applications include:

• Western Blot (WB)

• Indirect ELISA

• Reactivity with human samples

Researchers should always optimize these conditions for their specific experimental systems to obtain optimal results.

How should researchers optimize antigen retrieval for ARID4B immunohistochemistry?

Antigen retrieval is a critical step for successful ARID4B immunohistochemistry. Based on experimental validation:

The recommended primary protocol involves using TE buffer at pH 9.0 for antigen retrieval . This alkaline pH has been shown to effectively unmask ARID4B epitopes in formalin-fixed, paraffin-embedded tissues including pancreatic cancer, breast cancer, cervical cancer, and testis tissues.

As an alternative method, researchers can perform antigen retrieval using citrate buffer at pH 6.0 . When optimizing antigen retrieval:

• Compare both TE and citrate buffer methods side-by-side to determine which provides better signal-to-noise ratio

• Ensure appropriate incubation time (typically 15-20 minutes) at optimal temperature

• Include positive control tissues (such as testis or breast cancer samples) known to express ARID4B

• Run parallel negative controls (omitting primary antibody) to assess background staining

Optimization of antigen retrieval conditions is particularly important for tissues with high fixation variability or when working with archived samples.

What are the best practices for validating the specificity of ARID4B antibodies?

Ensuring ARID4B antibody specificity is crucial for generating reliable research data. Recommended validation approaches include:

• Molecular weight verification: ARID4B has a calculated molecular weight of 148 kDa (1312 amino acids), while observed bands typically appear at 100 kDa and 200 kDa . Discrepancies between calculated and observed weights may be due to post-translational modifications or protein processing.

• Knockdown/knockout controls: Utilizing ARID4B knockdown or knockout samples as negative controls. Published applications have validated antibody specificity using KD/KO systems .

• Multiple antibody approach: Compare results using different antibodies targeting distinct epitopes of ARID4B (such as comparing monoclonal and polyclonal antibodies).

• Recombinant protein controls: Use purified recombinant ARID4B protein as a positive control in Western blot experiments.

• Tissue/cell type controls: Include samples with known ARID4B expression levels. MCF-7 cells, A549 cells, and K-562 cells have been validated as positive controls for ARID4B expression .

• Cross-reactivity assessment: Test antibody reactivity against related proteins, particularly ARID4A which shares sequence similarity with ARID4B.

These validation steps should be performed before conducting critical experiments to ensure reliability of results and accurate interpretation of data.

How can researchers effectively study the role of ARID4B in cancer progression?

Investigating ARID4B's role in cancer requires a multifaceted approach:

• Expression analysis in cancer tissues: Compare ARID4B expression between tumor and matched normal tissues. ARID4B is highly expressed in breast, lung, colon, pancreatic, and ovarian cancers , suggesting potential oncogenic functions in these tissues.

• Mechanistic studies: Analyze ARID4B's interactions with key cancer-related pathways:

  • Examine its role in the Sin3A corepressor complex, where it may function in transcriptional repression

  • Investigate its involvement in cell cycle regulation and proliferation

  • Study its interaction with other transcription factors

• Functional studies using genetic manipulation:

  • Use RNA interference (siRNA/shRNA) to knock down ARID4B expression

  • Employ CRISPR-Cas9 to create ARID4B knockout cell lines

  • Perform rescue experiments with wild-type or mutant ARID4B

• Animal models: Develop ARID4B knockout or transgenic mouse models to study its role in tumorigenesis in vivo.

• Clinical correlation: Analyze ARID4B expression in patient samples and correlate with clinical outcomes, response to therapy, and other clinical parameters.

By integrating these approaches, researchers can comprehensively investigate ARID4B's functional roles in cancer initiation, progression, and potential as a therapeutic target.

What are the challenges in studying ARID4B protein-protein interactions, and how can they be addressed?

Studying ARID4B protein-protein interactions presents several challenges:

• Nuclear localization: ARID4B localizes to both nucleus and cytoplasm, with nuclear ARID4B being less stable due to rapid degradation by the ubiquitin-proteasome system . This differential stability complicates interaction studies.

• Technical challenges:

  • The large size of ARID4B (1312 amino acids) can make recombinant protein production difficult

  • Multiple protein isoforms may exhibit different interaction profiles

  • Transient or weak interactions may be missed by standard approaches

• Solutions and methodologies:

  • Optimized immunoprecipitation: Use crosslinking agents to stabilize transient interactions. The validated protocol recommends 0.5-4.0 μg of antibody for 1.0-3.0 mg of total protein lysate .

  • Proximity labeling approaches: BioID or APEX2 fusion proteins can identify proximal proteins regardless of interaction strength.

  • Mass spectrometry-based approaches: Analyze ARID4B interactome using quantitative proteomics after immunoprecipitation.

  • Subcellular fractionation: Separate nuclear and cytoplasmic fractions before performing interaction studies to account for compartment-specific interactions.

  • Domain-specific interaction mapping: Generate constructs expressing specific ARID4B domains to map domain-specific interactions.

• Verification approaches:

  • Confirm interactions using multiple techniques (co-IP, proximity ligation assay, FRET)

  • Validate physiological relevance through functional assays

  • Perform reciprocal IPs to strengthen evidence for interactions

These strategies can help overcome the inherent challenges of studying ARID4B's protein interaction network.

How does ARID4B differ from other ARID family members in its molecular functions?

ARID4B possesses distinct characteristics compared to other ARID family members:

• Structural differences:

  • Contains Tudor domains and N-terminal domain in retinoblastoma-binding protein-1 family domains, which are not present in all ARID family members

  • Lacks the LXCXE motif present in ARID4A, suggesting potentially different interactions with retinoblastoma protein

• Functional roles:

  • While many ARID family members (like ARID1A) act as tumor suppressors, ARID4B shows context-dependent functions

  • ARID4B may function in the assembly and/or enzymatic activity of the Sin3A corepressor complex

  • Unlike some ARID family members that are strictly nuclear, ARID4B localizes to both nucleus and cytoplasm

• Expression patterns:

  • ARID4B shows elevated expression in certain cancers (breast, lung, colon, pancreatic, ovarian), whereas other ARID family members like ARID1A often show reduced expression in cancers

  • ARID4B is highly expressed in testis, with lower expression in thymus, prostate, and ovary

• Cancer relevance:

  • While ARID1A mutations are frequent in multiple cancer types (endometrial, gastric, bladder), ARID4B mutations are less commonly reported

  • ARID4B may interact differently with the p16/RB tumor suppressor machinery compared to other family members

Understanding these differences is crucial for researchers targeting specific ARID family members and interpreting the results of ARID4B-focused experiments in the broader context of ARID family functions.

What are common issues when working with ARID4B antibodies and how can they be resolved?

Researchers frequently encounter several challenges when working with ARID4B antibodies:

• Multiple bands in Western blot:

  • ARID4B has observed molecular weights of 100 kDa and 200 kDa despite a calculated weight of 148 kDa

  • Resolution: Verify band identity through knockdown experiments, use appropriate positive controls, and optimize gel separation for high molecular weight proteins

• Weak or absent signal:

  • Potential causes: Low target expression, antibody degradation, or insufficient antigen retrieval

  • Resolution: Increase antibody concentration within recommended ranges (1:500-1:1000 for WB, 1:500-1:2000 for IHC) , optimize incubation time and temperature, and ensure proper sample preparation

• High background:

  • Potential causes: Non-specific binding, excessive antibody concentration, inadequate blocking

  • Resolution: Increase blocking time, optimize antibody dilution, use additional washing steps, and consider alternative blocking agents

• Variability between experiments:

  • Resolution: Standardize protocols, prepare fresh working solutions, aliquot antibodies to avoid freeze-thaw cycles, and use consistent positive controls

• Storage and stability issues:

  • The antibody should be stored at -20°C and is stable for one year after shipment

  • Aliquoting is unnecessary for -20°C storage for the polyclonal antibody

  • The monoclonal antibody requires storage at -80°C

Following these troubleshooting approaches can significantly improve experimental outcomes when working with ARID4B antibodies.

How can researchers optimize ARID4B antibody-based immunofluorescence experiments?

Successful immunofluorescence experiments with ARID4B antibodies require careful optimization:

• Cell line selection:

  • MCF-7 cells have been validated for ARID4B immunofluorescence and provide a reliable positive control

  • Consider cell lines with known ARID4B expression levels based on your research question

• Fixation and permeabilization:

  • ARID4B localizes to both nucleus and cytoplasm, requiring balanced fixation and permeabilization

  • Test both cross-linking (paraformaldehyde) and precipitating (methanol) fixatives

  • Optimize permeabilization conditions to ensure antibody accessibility to nuclear ARID4B

• Antibody concentration and incubation:

  • Start with the recommended dilution range (1:200-1:800) and optimize

  • Consider extending primary antibody incubation to overnight at 4°C to improve signal

  • Use fluorophore-conjugated secondary antibodies appropriate for your microscopy system

• Controls and validation:

  • Include positive and negative controls in each experiment

  • Consider co-staining with nuclear markers to verify subcellular localization

  • Perform ARID4B knockdown to confirm specificity of the signal

• Image acquisition and analysis:

  • Capture both nuclear and cytoplasmic signals due to ARID4B's dual localization

  • Consider z-stack imaging to fully capture the 3D distribution

  • Quantify nuclear vs. cytoplasmic distribution as this may have functional relevance

These optimization steps will enhance the quality and reliability of ARID4B immunofluorescence experiments, enabling better visualization of this protein's subcellular distribution.

What considerations should researchers take into account when selecting between monoclonal and polyclonal ARID4B antibodies?

The choice between monoclonal (67384-1-PBS) and polyclonal (24499-1-AP) ARID4B antibodies depends on specific experimental needs:

• Application compatibility:

  • The polyclonal antibody has been validated for WB, IP, IHC, IF/ICC, and ELISA

  • The monoclonal antibody has been validated for WB and Indirect ELISA

  • For applications like IHC or IF, the polyclonal antibody offers validated protocols

• Epitope recognition:

  • Polyclonal antibodies recognize multiple epitopes, potentially providing stronger signals

  • Monoclonal antibodies offer higher specificity for a single epitope, reducing background

• Experimental reproducibility:

  • Monoclonal antibodies provide consistent lot-to-lot reproducibility

  • Polyclonal antibodies may show batch variation but can be more robust against epitope changes

• Storage requirements:

  • The polyclonal antibody can be stored at -20°C and is stable for one year

  • The monoclonal antibody requires storage at -80°C

• Buffer compatibility:

  • The polyclonal antibody is supplied in PBS with 0.02% sodium azide and 50% glycerol

  • The monoclonal antibody is supplied in PBS only

  • Consider buffer components when planning experiments sensitive to these additives

• Research question considerations:

  • For detecting specific ARID4B isoforms or phosphorylation states, monoclonal antibodies may be preferred

  • For maximum sensitivity in detecting low abundance protein, polyclonal antibodies may provide advantages

Researchers should select the appropriate antibody based on these considerations and their specific experimental requirements.

How can researchers explore the role of ARID4B in epigenetic regulation?

ARID4B's involvement in epigenetic regulation offers promising research directions:

• Chromatin immunoprecipitation (ChIP) approaches:

  • Optimize ChIP protocols using ARID4B antibodies to identify genomic binding sites

  • Perform ChIP-seq to map ARID4B binding genome-wide

  • For IP applications with ARID4B antibody, use 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate

• Study ARID4B in the context of the Sin3A corepressor complex:

  • Investigate ARID4B's role in recruiting histone deacetylases (HDACs) to specific genomic loci

  • Analyze how ARID4B influences histone modification patterns at target genes

  • Examine how ARID4B mediates interactions between Sin3A and other regulatory complexes

• Transcriptional repression assays:

  • Use reporter assays to measure ARID4B's impact on gene expression

  • Analyze changes in the transcriptome after ARID4B depletion or overexpression

  • Investigate how ARID4B interacts with transcription factors and epigenetic machinery

• Cancer epigenetics:

  • Explore how ARID4B contributes to altered epigenetic landscapes in cancer

  • Investigate potential interactions with DNA methylation machinery

  • Examine ARID4B's role in maintaining cancer-specific gene expression patterns

• Single-cell approaches:

  • Apply single-cell technologies to understand cell-to-cell variation in ARID4B-mediated epigenetic regulation

  • Investigate how ARID4B contributes to epigenetic heterogeneity in tumors

These approaches will advance our understanding of ARID4B's role in epigenetic processes and potentially identify new therapeutic targets in diseases with dysregulated epigenetic pathways.

What potential exists for targeting ARID4B in cancer therapeutics?

Emerging research suggests several avenues for targeting ARID4B in cancer therapy:

• Expression patterns relevant to therapeutic development:

  • ARID4B is highly expressed in breast, lung, colon, pancreatic, and ovarian cancers

  • This expression pattern suggests potential value as a therapeutic target in these malignancies

• Potential therapeutic approaches:

  • Small molecule inhibitors: Design compounds that disrupt ARID4B's interaction with the Sin3A corepressor complex

  • Degradation-based approaches: Develop proteolysis-targeting chimeras (PROTACs) specific for ARID4B

  • Gene silencing strategies: Utilize siRNA or antisense oligonucleotides to reduce ARID4B expression

  • Functional antibodies: Develop antibodies that can modulate ARID4B function in vivo

• Biomarker potential:

  • Evaluate ARID4B expression as a predictive biomarker for response to epigenetic therapies

  • Correlate ARID4B levels with patient outcomes using validated antibodies for IHC (1:500-1:2000 dilution)

• Combination therapy approaches:

  • Investigate synergistic effects of ARID4B targeting with existing epigenetic drugs (HDAC inhibitors, DNA methyltransferase inhibitors)

  • Study how ARID4B inhibition affects sensitivity to conventional chemotherapeutics

• Target validation:

  • Use ARID4B antibodies to evaluate on-target effects of experimental therapeutics

  • Employ genetic approaches (CRISPR-Cas9) to validate ARID4B as a therapeutic target

  • Study compensatory mechanisms that may arise upon ARID4B inhibition

These approaches offer promising directions for translating ARID4B research into clinical applications, potentially expanding the repertoire of targeted therapies for cancers with ARID4B dysregulation.

What are the current limitations in ARID4B research and future research priorities?

Despite significant advances in understanding ARID4B function, several limitations and knowledge gaps remain:

• Technical limitations:

  • Current antibodies may not distinguish between ARID4B splice variants or post-translationally modified forms

  • Limited availability of highly specific inhibitors or activators for functional studies

  • Challenges in studying ARID4B in complex with other proteins due to its size and multi-domain structure

• Knowledge gaps:

  • Incomplete characterization of ARID4B genomic binding sites across different cell types

  • Limited understanding of how ARID4B contributes to tissue-specific gene regulation

  • Unclear mechanistic details of how ARID4B mediates interactions between different regulatory complexes

  • Insufficient data on ARID4B mutations and their functional consequences in different cancer types

• Future research priorities:

  • Develop more selective antibodies capable of distinguishing ARID4B isoforms and modifications

  • Expand ChIP-seq and other genomic approaches to map ARID4B binding sites across diverse cell types

  • Investigate ARID4B's role in normal development and tissue homeostasis

  • Explore therapeutic potential through development of specific inhibitors

  • Elucidate the structural basis of ARID4B interactions with DNA and protein partners

• Methodological advancements needed:

  • Improved protocols for studying chromatin-associated proteins in native complexes

  • Better animal models to study ARID4B function in development and disease

  • Advanced imaging techniques to visualize ARID4B dynamics in living cells

Addressing these limitations and priorities will advance our understanding of ARID4B biology and its potential therapeutic applications in cancer and other diseases.