ARP4A Antibody

What is the functional role of ARPC4/ARP4 proteins in cellular processes?

ARPC4 functions as an actin-binding component of the Arp2/3 complex, a multiprotein complex that mediates actin polymerization upon stimulation by nucleation-promoting factors. This complex is crucial for the formation of branched actin networks in the cytoplasm, which provides the force necessary for cell motility. Beyond its cytoplasmic role, the Arp2/3 complex containing ARPC4 also promotes actin polymerization in the nucleus, thereby regulating gene transcription and facilitating the repair of damaged DNA. Particularly noteworthy is its role in promoting homologous recombination (HR) repair in response to DNA damage by driving the motility of double-strand breaks (DSBs) .

In contrast, the similarly named but functionally distinct ARP4/ANGPTL4 (not to be confused with ARPC4) is a secreted protein expressed in adipose tissue, liver, and placenta. This protein plays varied roles in adipogenesis, angiogenesis (independent of VEGF), and carcinogenesis, with its expression observed in hypoxic human tissues and various cancers including liposarcoma, hepatocellular carcinoma, and renal cell carcinoma .

How do I determine which antibody format (polyclonal vs. monoclonal) is most appropriate for my ARPC4/Arp4 research?

When selecting between polyclonal and monoclonal antibodies for ARPC4/Arp4 research, consider your experimental objectives and required specificity. Polyclonal antibodies, such as the rabbit polyclonal ARPC4 antibody described in the literature, recognize multiple epitopes and provide robust detection across applications like immunoprecipitation (IP) and Western blotting (WB) . This multi-epitope recognition makes them advantageous for detecting proteins expressed at low levels or in applications requiring strong signal amplification.

The decision should be guided by your specific experimental needs, target localization, and whether you require detection of multiple conformational states of the protein or absolute epitope specificity.

What species cross-reactivity can I expect from commercially available ARPC4/ARP4 antibodies?

Based on the available data, commercial ARPC4 antibodies typically demonstrate reliable cross-reactivity with human and mouse samples . For example, the rabbit polyclonal ARPC4 antibody (ab217065) has been validated for both human and mouse samples in immunoprecipitation and Western blot applications. This cross-reactivity stems from the high conservation of actin-related proteins across mammalian species.

For highly specialized research involving non-mammalian models such as yeast Arp4p (used in kinetochore assembly studies), species-specific antibodies may be required, as seen in studies utilizing Flag-tagged Arp4p for ChIP chip analysis in yeast models .

What are the optimal sample preparation methods for detecting ARPC4/ARP4 in different subcellular compartments?

When investigating nuclear functions of ARPC4, consider:

• Nuclear-cytoplasmic fractionation before immunodetection

• DNase or sonication treatment to release chromatin-bound proteins

• Special fixation protocols for immunofluorescence that maintain nuclear architecture

For ChIP applications studying DNA-protein interactions, protocols similar to those used with Flag-tagged Arp4p in yeast studies can be adapted . These typically involve crosslinking with formaldehyde followed by chromatin fragmentation and immunoprecipitation.

If examining both cytoplasmic and nuclear pools simultaneously, sequential extraction protocols can provide compartment-specific enrichment while maintaining protein integrity and native conformation for optimal antibody recognition.

How should I design ChIP-chip or ChIP-seq experiments using ARPC4/ARP4 antibodies?

Successful ChIP-chip or ChIP-seq experiments with ARPC4/ARP4 antibodies require careful experimental design and optimization. Based on established protocols in the literature, consider the following methodology:

• Antibody selection and validation:

  • Verify the antibody's specificity for immunoprecipitation applications

  • Perform pilot ChIP experiments with positive and negative control regions

  • Consider epitope-tagged approaches (e.g., Flag-tagged ARPC4) if antibody performance is suboptimal

• Crosslinking and chromatin preparation:

  • Optimize formaldehyde crosslinking time (typically 10-15 minutes)

  • Ensure chromatin is sheared to appropriate fragment sizes (200-500bp)

  • Include input controls and non-specific antibody controls

• Immunoprecipitation conditions:

  • Determine optimal antibody concentration through titration experiments

  • Adjust washing stringency to minimize background while maintaining signal

  • Consider dual crosslinking approaches if standard protocols yield insufficient enrichment

• Data analysis considerations:

  • Apply appropriate normalization methods for platform-specific biases

  • Use software like Genome Shovel for high-resolution tiling array analysis

  • Deposit raw data in repositories like Gene Expression Omnibus for reproducibility and transparency

When designing these experiments, remember that nuclear actin-related proteins like ARPC4 can function in both gene transcription regulation and DNA repair processes , so experimental timing and cellular conditions should be chosen based on which functional aspect you're investigating.

What immunohistochemistry protocols are most effective for detecting ARPC4/ARP4 in tissue samples?

For effective immunohistochemical detection of actin-related proteins in tissue samples, a protocol similar to that used for ARPC1A (a related Arp2/3 complex subunit) can be adapted. Based on established methodologies, the following procedure is recommended:

• Tissue preparation and antigen retrieval:

  • Deparaffinize and rehydrate tissue sections thoroughly

  • Quench endogenous peroxidase activity with 0.3% H₂O₂ treatment for 30 minutes

  • Perform heat-induced epitope retrieval using sodium citrate buffer (10 mmol/L, pH 6.0) in a pressure cooker for 7 minutes

• Blocking and antibody incubation:

  • Block non-specific binding with 5% normal goat serum

  • Incubate with primary anti-ARPC4 antibody at optimized dilution (typically 1:200) overnight at 4°C

  • Apply appropriate species-specific secondary antibody (e.g., anti-rabbit for rabbit primary antibodies)

• Signal development and quantification:

  • Visualize using diaminobenzidine (DAB) as the chromogen

  • Counterstain with hematoxylin for nuclear visualization

  • Implement semi-quantitative scoring based on staining intensity (0-3) and proportion of positive cells (0-4)

For tissue microarray (TMA) applications, this protocol has been successfully employed to assess protein expression levels across multiple patient samples simultaneously, allowing for correlation with clinical parameters and outcomes. When investigating potential prognostic biomarkers, consistent staining conditions across all samples are essential for valid comparisons.

How can I troubleshoot weak or absent signals when using ARPC4/ARP4 antibodies in Western blotting?

When encountering weak or absent signals in Western blotting with ARPC4/ARP4 antibodies, systematic troubleshooting approaches should address several potential issues:

• Protein extraction efficiency:

  • ARPC4 functions in both cytoplasmic and nuclear compartments , so ensure your lysis buffer can effectively extract proteins from both locations

  • Consider using specialized nuclear extraction protocols if targeting nuclear ARPC4

  • Add protease inhibitors freshly to prevent degradation

• Antibody-specific considerations:

  • Verify the antibody recognizes your species of interest (validated for human and mouse samples)

  • Check if the antibody recognizes denatured epitopes (some antibodies only work in native conditions)

  • Optimize primary antibody concentration through titration experiments

  • Extend primary antibody incubation time (overnight at 4°C)

• Technical optimizations:

  • Increase protein loading (20-50 μg per lane)

  • Reduce membrane blocking stringency (try 3% BSA instead of 5%)

  • Use enhanced chemiluminescence (ECL) substrates with higher sensitivity

  • Consider specialized membrane types (PVDF often provides better protein retention than nitrocellulose)

• Sample preparation refinements:

  • Adjust reducing conditions (fresh DTT or β-mercaptoethanol)

  • Optimize SDS-PAGE conditions for proteins in the 20 kDa range (ARPC4 size)

  • Reduce transfer time or voltage for small proteins to prevent over-transfer

If problems persist after these optimizations, consider validating antibody functionality using positive control lysates from cells known to express high levels of ARPC4/ARP4 or recombinant protein standards.

How should I interpret discrepancies between antibody-based detection methods for ARPC4/ARP4?

Discrepancies between different antibody-based detection methods for ARPC4/ARP4 require careful analysis to determine whether they represent technical artifacts or biologically meaningful differences in protein behavior.

When encountering such discrepancies, consider the following interpretive framework:

• Method-specific protein accessibility:

  • In immunohistochemistry, formalin fixation may mask epitopes that are readily accessible in Western blotting

  • Nuclear localization of ARPC4 might be difficult to detect with certain antibodies due to chromatin compaction

  • Different solubilization methods might extract distinct protein pools

• Protein complex integrity:

  • ARPC4 functions as part of the Arp2/3 complex , and some antibodies may preferentially recognize free versus complex-bound forms

  • Co-immunoprecipitation results may reflect indirect interactions within larger multiprotein assemblies

• Post-translational modifications:

  • Discrepancies might reflect differential detection of modified protein forms

  • Phosphorylation or other modifications might affect epitope accessibility in a method-dependent manner

• Technical considerations:

  • Compare antibody clones used across methods (different clones recognize different epitopes)

  • Evaluate detection thresholds (Western blotting might be more sensitive than immunohistochemistry)

  • Assess quantification methods (band intensity versus staining score systems)

When publishing results with such discrepancies, transparently report all methodological details and consider validating key findings with multiple antibodies or orthogonal techniques such as mass spectrometry or gene expression analysis.

What controls are essential when validating new ARPC4/ARP4 antibodies for research applications?

Rigorous validation of new ARPC4/ARP4 antibodies requires a comprehensive set of controls to ensure specificity, sensitivity, and reproducibility. Essential controls include:

• Positive and negative cell/tissue controls:

  • Cell lines with documented high expression (positive control)

  • Cell lines with confirmed low/no expression (negative control)

  • Ideally, include knockout or knockdown samples as definitive negative controls

  • Tissues with known expression patterns based on mRNA data

• Antigen competition assays:

  • Pre-incubate antibody with excess purified antigen peptide

  • Signal should be eliminated or significantly reduced in antigen-competed samples

  • Particularly important for antibodies raised against synthetic peptides

• Molecular weight verification:

  • Confirm detection at the expected molecular weight (approximately 20 kDa for ARPC4)

  • Assess potential detection of isoforms or processed forms

  • Compare with alternative antibodies targeting different epitopes of the same protein

• Cross-reactivity assessment:

  • Test against closely related family members (other Arp2/3 complex subunits)

  • Evaluate species cross-reactivity if intended for multi-organism studies

  • Document any nonspecific binding patterns

• Application-specific controls:

  • For immunoprecipitation: perform reverse IP and test in IgG control samples

  • For immunohistochemistry: include isotype controls and absorption controls

  • For ChIP: include input controls and unrelated antibody controls

This systematic validation approach aligns with current best practices in the antibody field and will help researchers avoid misleading results from insufficiently characterized reagents.

How can I use ARPC4/ARP4 antibodies to study the relationship between actin dynamics and DNA repair mechanisms?

ARPC4/ARP4 antibodies provide powerful tools for investigating the emerging relationship between actin dynamics and DNA repair mechanisms. Recent research has revealed that the Arp2/3 complex, which includes ARPC4, plays crucial roles in nuclear actin polymerization that drives homologous recombination (HR) repair following DNA damage .

To study these relationships:

• Chromatin immunoprecipitation approaches:

  • Perform ChIP experiments following DNA damage induction (e.g., with ionizing radiation or etoposide)

  • Use sequential ChIP (re-ChIP) to identify co-localization with DNA repair factors

  • Couple with high-resolution genomic approaches like ChIP-seq to identify genome-wide binding patterns

• Proximity ligation assays (PLA):

  • Employ PLA to detect in situ interactions between ARPC4 and DNA repair proteins

  • Visualize spatial relationships at sites of DNA damage

  • Quantify interaction frequencies under different damage conditions

• Live-cell imaging with complementary markers:

  • Combine immunofluorescence for ARPC4 with markers of DNA damage (γH2AX)

  • Track recruitment kinetics following localized DNA damage

  • Correlate actin dynamics with repair progression

• Functional studies with domain-specific antibodies:

  • Use antibodies targeting different ARPC4 domains to block specific interactions

  • Microinject function-blocking antibodies to assess acute effects on repair

  • Combine with nuclease inhibitors to dissect mechanism dependencies

This approach can help elucidate how actin-related proteins facilitate the mobility of double-strand breaks during repair processes , potentially leading to new therapeutic strategies targeting this mechanism in cancer cells.

What approaches can I use to study ARPC4/ARP4's role in cancer progression and metastasis?

Investigating ARPC4/ARP4's role in cancer progression and metastasis requires multi-faceted approaches that combine antibody-based detection with functional studies. Building on research demonstrating the prognostic significance of related actin complex proteins in cancers , consider these methodological approaches:

• Tissue-based expression analysis:

  • Perform immunohistochemistry on tissue microarrays containing primary tumors and matched metastases

  • Implement semi-quantitative scoring systems to assess expression levels

  • Correlate expression with clinicopathological features and patient outcomes

  • Use multivariate analysis to establish independent prognostic value

• Mechanistic studies in cancer models:

  • Evaluate ARPC4 expression across cancer cell lines with varying metastatic potential

  • Perform knockdown/knockout studies followed by migration, invasion, and 3D culture assays

  • Use antibodies to monitor changes in ARPC4 localization during epithelial-mesenchymal transition

  • Assess co-localization with invasive structures like invadopodia

• In vivo metastasis models:

  • Generate stable ARPC4-modulated cell lines for xenograft studies

  • Use antibodies to track ARPC4 expression in circulating tumor cells

  • Analyze ARPC4 in primary tumors versus metastatic sites

• Multi-omics integration:

  • Correlate antibody-based protein detection with transcriptomic profiles

  • Apply network analysis approaches like Weighted Gene Co-expression Network Analysis (WGCNA) to identify ARPC4-associated gene modules

  • Validate key interactions using co-immunoprecipitation with ARPC4 antibodies

These approaches can help determine whether ARPC4 holds similar prognostic value to ARPC1A, which has been identified as a biomarker in prostate cancer , and may reveal therapeutic vulnerabilities in actin regulatory pathways.

How can computational models enhance the selection and application of antibodies for ARPC4/ARP4 research?

Computational models offer powerful approaches to enhance antibody selection and application in ARPC4/ARP4 research, particularly when dealing with complex specificity requirements or cross-reactivity concerns. These advanced approaches include:

• Epitope prediction and antibody design:

  • Implement biophysics-informed modeling to identify optimal epitopes unique to ARPC4

  • Design antibodies with customized specificity profiles through computational approaches

  • Distinguish between binding modes associated with particular ligands or epitopes

  • Computationally optimize antibody sequences for enhanced specificity or cross-reactivity

• High-throughput sequence analysis:

  • Apply deep sequencing analysis to phage display selections to identify antibody variants with desired binding properties

  • Identify binding modes that can distinguish between closely related epitopes

  • Use computational methods to disentangle binding profiles even when target epitopes are chemically similar

• Structure-based optimization:

  • Implement molecular dynamics simulations to predict antibody-antigen interactions

  • Model conformational changes in ARPC4 that might affect epitope accessibility

  • Design antibodies that can distinguish between free ARPC4 and complex-incorporated forms

• Cross-reactivity prediction:

  • Apply machine learning algorithms to predict potential cross-reactivity with related actin complex proteins

  • Identify sequence regions unique to ARPC4 versus other ARP family members

  • Optimize antibody design to minimize unwanted interactions

These computational approaches, combined with experimental validation, can generate antibodies with either highly specific binding to ARPC4 alone or controlled cross-reactivity when studying multiple actin-related proteins simultaneously .

How can I distinguish between ARPC4 and other actin-related proteins when using antibodies?

Distinguishing between ARPC4 and other actin-related proteins requires careful antibody selection and validation strategies:

• Epitope selection considerations:

  • Choose antibodies targeting unique regions of ARPC4 not conserved in other ARP family members

  • Avoid antibodies raised against highly conserved actin-binding domains

  • Review sequence alignments between ARPC4 and potentially cross-reactive proteins (ARPC1A, etc.)

  • Verify that antibodies target unique epitopes (e.g., within Human ARPC4 aa 1-50)

• Validation using genetic approaches:

  • Implement siRNA/shRNA knockdown of ARPC4 specifically

  • Utilize CRISPR/Cas9-mediated knockout cells as definitive negative controls

  • Perform rescue experiments with exogenous ARPC4 expression to confirm specificity

• Biochemical differentiation:

  • Use size-based differentiation (ARPC4: ~20kDa vs. other ARPs of different molecular weights)

  • Perform two-dimensional gel electrophoresis to separate based on both size and isoelectric point

  • Implement sequential immunoprecipitation to discriminate between protein complexes

• Functional validation:

  • Correlate antibody signals with known subcellular distributions of specific ARPs

  • ARPC4 functions in both cytoplasmic actin networks and nuclear processes

  • Other ARPs may have distinct localization patterns and functional associations

When working with closely related proteins like ARPC1A and ARPC4, always validate findings with multiple antibodies and complementary techniques such as mass spectrometry for definitive protein identification.

What are the considerations for using anti-ARPC4/ARP4 antibodies in multiplexed immunoassays?

Implementing anti-ARPC4/ARP4 antibodies in multiplexed immunoassays requires careful consideration of several technical and biological factors:

• Antibody compatibility:

  • Select antibodies raised in different host species to enable simultaneous detection

  • Ensure primary antibodies can be distinguished by species-specific secondary antibodies

  • Consider directly conjugated antibodies to eliminate secondary antibody cross-reactivity

  • Validate that antibody performance is maintained in multiplexed conditions

• Signal separation strategies:

  • Implement spectral unmixing for fluorescent detection systems

  • Use brightfield multiplexing with distinct chromogens for immunohistochemistry

  • Consider sequential detection protocols with intermittent stripping or quenching

  • Validate signal specificity with single-stain controls

• Epitope accessibility:

  • Evaluate whether detection of multiple targets requires different antigen retrieval methods

  • Determine optimal fixation conditions compatible with all target epitopes

  • Test order-of-addition effects when detecting multiple actin-related proteins

• Quantitative considerations:

  • Establish detection thresholds for each antibody in the multiplex panel

  • Implement standardized scoring systems for consistent evaluation

  • Use digital image analysis algorithms to quantify multiple markers objectively

  • Include appropriate calibration standards for each target

These considerations become particularly important when studying ARPC4 alongside other Arp2/3 complex components or when examining relationships between actin-related proteins and cancer biomarkers in clinical specimens.

How can synthetic peptide arrays be used to map epitopes recognized by ARPC4/ARP4 antibodies?

Synthetic peptide arrays provide powerful tools for precise epitope mapping of ARPC4/ARP4 antibodies, enabling enhanced characterization and validation:

• Array design strategy:

  • Generate overlapping peptides (typically 12-20 amino acids) spanning the entire ARPC4 sequence

  • Include alanine-substitution variants to identify critical binding residues

  • Incorporate known post-translational modification sites as modified peptides

  • Design control peptides from related ARP family members to assess cross-reactivity

• Implementation methodology:

  • Synthesize peptides directly on array surfaces or spot pre-synthesized peptides

  • Incubate arrays with antibodies at optimized concentrations

  • Detect binding using labeled secondary antibodies or direct detection systems

  • Analyze binding patterns to identify core epitope regions

• Data interpretation:

  • Define the minimal epitope sequence required for antibody recognition

  • Identify key residues by analyzing effects of alanine substitutions

  • Assess effects of post-translational modifications on antibody binding

  • Compare epitope accessibility in the context of protein structure models

• Application to antibody selection:

  • Choose antibodies recognizing epitopes in functionally relevant domains

  • Select antibodies with epitopes that remain accessible in the Arp2/3 complex

  • Identify epitopes unique to ARPC4 versus related proteins

  • Design custom antibodies against identified specific epitopes

This approach is particularly valuable for antibodies targeting synthetic peptide immunogens (such as those within Human ARPC4 aa 1-50) , as it can precisely define the recognized epitope and predict potential cross-reactivity with related proteins or affected by structural changes during protein functions.