ARP4 Antibody

What is ARP4 and why are antibodies against it important in research?

ARP4 refers to two distinct proteins that share the same abbreviation in scientific literature. The first is actin-related protein 4 (Arp4), a nuclear protein that functions as a novel suppressor for nuclear F-actin formation in mammalian cells. It is included in multiple chromatin remodeling complexes and directly binds to G-actin, inhibiting actin polymerization . The second is angiopoietin-related protein 4 (ANGPTL4), which mediates inactivation of lipoprotein lipase and plays roles in triglyceride clearance, lipid metabolism, and angiogenesis .

Antibodies against these proteins are critical research tools because they enable scientists to detect, quantify, localize, and study the functions of these proteins in various biological contexts. For nuclear Arp4, antibodies help investigate chromatin remodeling mechanisms, while for ANGPTL4/ARP4, they assist in studying lipid metabolism, angiogenesis, and cancer progression .

How do I determine which ARP4 antibody is appropriate for my specific research application?

To select the appropriate ARP4 antibody, first clarify which ARP4 protein you're studying (nuclear Arp4 or ANGPTL4). Then consider these factors:

• Experimental technique: Different applications require antibodies with specific characteristics:

  • For Western blotting: Select antibodies validated for this application with known band patterns

  • For immunofluorescence: Choose antibodies that recognize native protein conformations

  • For ChIP experiments: Use antibodies specifically validated for chromatin immunoprecipitation

• Species compatibility: Ensure the antibody recognizes your species of interest. For example, if studying human BAF53 (human Arp4 homolog), select an antibody specific to human proteins .

• Validation evidence: Review all available validation data including:

  • Knockdown/knockout controls

  • Recombinant protein controls

  • Cross-reactivity assessments

As the antibody characterization crisis has shown, approximately 50% of commercial antibodies fail to meet basic standards for characterization, resulting in financial losses of $0.4–1.8 billion per year in the United States alone . Therefore, proper antibody selection is crucial for research reliability.

What validation methods should I use to confirm ARP4 antibody specificity?

Comprehensive validation of ARP4 antibodies is essential to ensure experimental reliability. Implement these validation approaches:

• Genetic validation:

  • Use Arp4 knockdown cells (Arp4 KD) as negative controls

  • Compare signals between wild-type and knockout/knockdown samples

  • Generate an Arp4 mutant (e.g., arp4S23A/D159A) to confirm specificity

• Biochemical validation:

  • Western blot analysis with recombinant ARP4 protein

  • Peptide competition assays to confirm epitope specificity

  • Immunoprecipitation followed by mass spectrometry

• Cross-platform validation:

  • Compare results across multiple techniques (Western blot, immunofluorescence, ChIP)

  • Use orthogonal methods to detect the protein (e.g., tagged protein expression)

• Application-specific controls:

  • For ChIP experiments: Include IgG controls and known target regions

  • For immunofluorescence: Include peptide blocking controls

Remember that validation should be performed for each specific application, as an antibody validated for Western blot may not work for immunofluorescence or ChIP .

How can I optimize immunoprecipitation protocols using ARP4 antibodies for chromatin studies?

Optimizing immunoprecipitation (IP) with ARP4 antibodies for chromatin studies requires careful attention to several factors:

• Chromatin preparation:

  • For nuclear Arp4 studies, optimize crosslinking conditions (typically 1% formaldehyde for 10 minutes)

  • Ensure proper chromatin fragmentation (200-500bp fragments)

  • Verify nuclear extraction efficiency before proceeding

• Antibody selection and incubation:

  • Use monoclonal antibodies for higher specificity (like those available through DSHB)

  • Test different antibody concentrations (typically 2-10μg per IP)

  • Optimize incubation time and temperature (4°C overnight is standard)

• Washing conditions:

  • Design washing buffers with appropriate stringency

  • Include controls to detect non-specific binding

• Elution and analysis:

  • For ChIP-chip analysis of Arp4, follow protocols similar to those used for Flag-tagged Arp4p

  • When analyzing results, use appropriate software like Genome Shovel for visualization

Researchers studying the SWR1 complex (which contains Arp4) should particularly focus on optimizing conditions that preserve protein complex integrity while ensuring specificity of the pull-down .

How can I use ARP4 antibodies to investigate nuclear F-actin formation and regulation?

ARP4 antibodies provide powerful tools for investigating nuclear F-actin formation and regulation. Follow these methodological approaches:

• Comparative analysis using knockdown models:

  • Generate Arp4 knockdown (Arp4 KD) cells as demonstrated in previous studies

  • Use Arp4 antibodies to confirm knockdown efficiency via Western blot

  • Compare nuclear F-actin formation between control and Arp4 KD cells using fluorescence microscopy

• Co-localization studies:

  • Perform dual immunofluorescence with Arp4 antibodies and fluorescently labeled phalloidin to visualize F-actin

  • Quantify the intensity of nuclear F-actin and correlate with Arp4 levels

  • Use high-resolution microscopy techniques like STORM or STED for detailed visualization

• Mechanistic investigations:

  • Combine Arp4 antibodies with antibodies against other nuclear actin regulators

  • Investigate F-actin-inducible gene expression (e.g., OCT4) in relation to Arp4 levels

  • Analyze DNA damage repair efficiency in relation to Arp4-regulated nuclear F-actin formation

• In vitro reconstitution:

  • Use purified Arp4 and antibodies to study direct inhibition of F-actin formation

  • Examine the effects of Arp4 on actin dynamics using techniques like TIRF microscopy

Remember that "nuclear F-actin bundles were thickened by Arp4 KD" and "the intensity of nuclear F-actin was increased by Arp4 KD in a statistically significant manner," highlighting Arp4's role as a negative regulator of nuclear F-actin formation .

What approaches can I use to study ARP4's interactions with chromatin remodeling complexes?

Studying Arp4's interactions with chromatin remodeling complexes requires sophisticated techniques and careful experimental design:

• Co-immunoprecipitation strategies:

  • Use antibodies against Arp4 to pull down associated complex members

  • Alternatively, use antibodies against known complex components (e.g., BRG1, a part of the SWI/SNF chromatin remodeling complex) to co-precipitate Arp4

  • Confirm interactions via Western blot or mass spectrometry

• Chromatin localization analysis:

  • Perform ChIP-seq or ChIP-chip using Arp4 antibodies to map genomic binding sites

  • Follow protocols similar to those used for Flag-tagged Arp4p detection

  • Compare Arp4 binding profiles with those of other complex members

• Functional complex analysis:

  • Conduct chromatin remodeling assays in the presence of Arp4 antibodies to assess functional inhibition

  • Compare chromatin accessibility in normal versus Arp4-depleted cells using techniques like ATAC-seq

  • Study the effects of Arp4 on histone modifications through the NuA4 histone acetyltransferase complex

• Structural studies:

  • Use epitope-mapped antibodies to probe accessible regions of Arp4 within complexes

  • Combine with cryo-EM or crystallography data to understand complex architecture

These approaches will help elucidate Arp4's roles in multiple complexes, including the INO80 and SWR1 chromatin remodeling complexes and the NuA4 histone acetyltransferase complex .

Why might I observe discrepancies between different ARP4 antibodies in my experiments?

Discrepancies between different ARP4 antibodies are a common challenge and can be attributed to several factors:

• Epitope differences:

  • Different antibodies recognize distinct regions of the protein

  • Some epitopes may be masked in certain protein conformations or complexes

  • Post-translational modifications may affect epitope accessibility

• Antibody quality and validation:

  • As noted in the literature, approximately 50% of commercial antibodies fail to meet basic characterization standards

  • Inadequate validation in the specific application you're using

  • Batch-to-batch variability in antibody production

• Target protein complexity:

  • Confusion between the two different proteins both called "ARP4" (nuclear Arp4 vs. ANGPTL4/ARP4)

  • Detection of different isoforms or splice variants

  • Presence of Arp4 in different protein complexes that may mask epitopes

• Experimental conditions:

  • Different fixation methods affecting epitope preservation

  • Buffer compositions that influence antibody-antigen interactions

  • Sample preparation methods that alter protein conformation

To address these discrepancies, perform side-by-side comparisons under identical conditions, validate each antibody with appropriate controls, and carefully document the performance characteristics of each antibody in your specific experimental system.

How should I interpret Western blot results when using ARP4 antibodies?

Interpreting Western blot results with ARP4 antibodies requires careful analysis and consideration of several factors:

• Band size interpretation:

  • Nuclear Arp4: Expected molecular weight approximately 48-50 kDa

  • ANGPTL4/ARP4: Full-length protein ~45-50 kDa; cleaved N-terminal domain ~26 kDa

  • Consider post-translational modifications that may alter apparent molecular weight

• Multiple band analysis:

  • Multiple bands may represent:

    • Proteolytic fragments (especially for ANGPTL4, which undergoes cleavage)

    • Different isoforms or splice variants

    • Cross-reactivity with related proteins

  • Validate unexpected bands with knockdown/knockout controls

• Quantification considerations:

  • Use appropriate loading controls (e.g., GAPDH, β-actin)

  • Perform densitometry analysis with proper background subtraction

  • Ensure signal is within the linear range of detection

• Sample-specific factors:

  • Nuclear/cytoplasmic fractionation efficiency

  • Protein extraction method compatibility

  • Sample buffer composition and heating conditions

When comparing results with literature, note that different antibodies may detect different forms of the protein. For instance, some antibodies may preferentially recognize the N-terminal domain of ANGPTL4/ARP4, which "has higher activity in LPL inactivation than the uncleaved protein" .

How can I use ARP4 antibodies to investigate cancer biology, particularly in renal carcinoma?

ARP4 antibodies offer valuable tools for investigating cancer biology, with particularly relevant applications in renal carcinoma research:

• Diagnostic and prognostic applications:

  • Examine ANGPTL4/ARP4 expression levels in tumor versus normal tissue

  • Correlate expression with clinical outcomes and treatment response

  • ARP4 mRNA expression is elevated in clear cell renal carcinoma compared to adjacent non-tumor tissue

• Mechanistic studies:

  • Investigate the dual role of ANGPTL4/ARP4 as:

    • A proangiogenic factor for endothelial cells

    • A survival factor for renal carcinoma cells

  • Study the effects of ARP4 (500ng/ml) on the survival of 786-0 renal carcinoma cells

• Therapeutic development:

  • Test neutralizing antibodies against ANGPTL4/ARP4 in cancer models

  • The fully human monoclonal antibody CR064 inhibits both ARP4-mediated angiogenesis and survival of renal carcinoma cells in vitro

  • Evaluate antibody efficacy using migration and tube formation assays

• Experimental approaches:

  • Use immunohistochemistry with ARP4 antibodies to assess tumor vascularization

  • Perform functional assays with recombinant ARP4 and neutralizing antibodies

  • ARP4 (250ng/ml) enhances HUVEC migration approximately 2-fold and induces tube formation in matrigel

The table below summarizes key functional effects of ARP4 and inhibition by CR064 antibody:

These findings support investigating neutralizing antibodies against ANGPTL4/ARP4 as potential therapeutic approaches for renal cell carcinoma .

What methods can I use to study ARP4's role in chromatin dynamics and gene expression?

To investigate ARP4's role in chromatin dynamics and gene expression, implement these advanced methodological approaches:

• Genome-wide binding analysis:

  • Perform ChIP-seq or ChIP-chip using ARP4 antibodies

    • Tag Arp4p at the carboxy terminus with the Flag epitope for detection with anti-FLAG M2 antibody

    • Follow immunoprecipitation and DNA amplification protocols as described in the literature

    • Analyze data using appropriate software like Genome Shovel

  • Compare binding profiles with other chromatin remodeling complex components

• Transcriptional impact studies:

  • Analyze transcriptional changes in Arp4 knockdown or mutant cells

  • Compare with transcriptional profiles of cells with alterations in other complex components

  • Note that "most of the genome-wide transcriptional defects seen in swr1 cells are also found in htz1 cells"

• Chromatin accessibility analysis:

  • Combine Arp4 antibody ChIP with nuclease accessibility assays

  • Perform ATAC-seq in Arp4-depleted versus control cells

  • Map changes in chromatin structure to Arp4 binding sites

• Functional domain studies:

  • Use Arp4 mutants (e.g., arp4S23A/D159A) to identify functional domains

  • Correlate structure-function relationships with chromatin binding patterns

  • Investigate the role of specific domains in protein-protein interactions

• Dynamic association analysis:

  • Employ live-cell imaging techniques with fluorescently tagged Arp4

  • Use FRAP (Fluorescence Recovery After Photobleaching) to study dynamic binding

  • Validate findings with immunofluorescence using Arp4 antibodies

These approaches will help elucidate how Arp4, as part of chromatin remodeling complexes, influences gene expression and chromatin structure at the genome-wide level.

How can I use ARP4 antibodies to investigate the relationship between nuclear actin dynamics and gene regulation?

Investigating the relationship between nuclear actin dynamics and gene regulation using ARP4 antibodies requires sophisticated experimental approaches:

• Gene expression correlation studies:

  • Use RT-qPCR to analyze expression of F-actin-inducible genes (e.g., OCT4) in Arp4 KD cells

  • Compare gene expression patterns between control and Arp4-depleted cells

  • Correlate changes in nuclear F-actin levels with transcriptional alterations

• Chromatin immunoprecipitation approaches:

  • Perform sequential ChIP (ChIP-reChIP) with antibodies against Arp4 and RNA polymerase II

  • Identify genomic regions where Arp4-regulated actin dynamics influence transcription

  • Combine with RNA-seq to correlate binding with expression changes

• Live-cell visualization techniques:

  • Implement dual labeling with Arp4 antibodies and actin probes

  • Use super-resolution microscopy to visualize nuclear actin structures

  • Correlate dynamic changes in nuclear F-actin with transcriptional activity

• Mechanistic intervention approaches:

  • Treat cells with actin polymerization modulators while monitoring Arp4 localization

  • Perform experiments in NIH3T3 cells, where "nuclear F-actin bundles were thickened by Arp4 KD"

  • Use mouse nuclei transplanted into Xenopus laevis oocytes to study the inhibitory effects of purified Arp4 on F-actin formation

The central finding that "Arp4 has a critical role in the formation and functions of nuclear F-actin" provides a foundation for exploring how nuclear actin dynamics regulated by Arp4 influence gene expression in different cellular contexts.

What considerations are important when developing new antibodies against ARP4 for research applications?

Developing new antibodies against ARP4 requires careful planning and validation strategies to avoid common pitfalls in antibody production:

• Antigen design considerations:

  • Select unique, accessible epitopes to distinguish between:

    • Nuclear Arp4 versus cytoplasmic actin or other actin-related proteins

    • ANGPTL4/ARP4 versus other angiopoietin family members

  • Consider recombinant protein versus synthetic peptide approaches

  • Evaluate potential post-translational modifications that might affect epitope recognition

• Validation requirements:

  • Implement comprehensive validation strategies to address the "antibody characterization crisis"

  • Follow standards like those established by NeuroMab, which performs:

    • Immunohistochemistry and Western Blots

    • Testing in knockout models

    • Multiple application-specific validation steps

  • Document outcomes of both positive and negative evaluations transparently

• Production methodology:

  • Consider developing recombinant antibodies with defined sequences

    • Make DNA sequences and plasmids readily available through non-profit, open-access sources

    • Consider converting monoclonal antibodies to recombinant formats for improved reproducibility

  • For monoclonal antibodies:

    • Establish stable hybridomas with consistent production characteristics

    • Sequence VH and VL regions to enable future conversion to recombinant formats

• Application-specific optimization:

  • Optimize antibodies for specific applications (Western blot, immunofluorescence, ChIP)

  • Provide detailed protocols for each application to enhance reproducibility

  • Document limitations and optimal conditions for each application

Following these principles will help address the estimated $0.4–1.8 billion yearly losses due to poorly characterized antibodies and contribute to more reliable research tools for the scientific community.