ARPC2A Antibody

What is ARPC2 and what is its functional significance in cellular processes?

ARPC2 (Actin-related protein 2/3 complex subunit 2) is a critical component of the Arp2/3 complex that mediates actin polymerization when stimulated by nucleation-promoting factors (NPFs). This 34.3 kDa protein (300 amino acids in length) functions as an actin-binding component that contacts the mother actin filament and facilitates the formation of branched actin networks in the cytoplasm . Its primary function is to provide the mechanical force necessary for cell motility through cytoskeletal reorganization. Beyond its cytoplasmic role, ARPC2 also promotes actin polymerization in the nucleus, where it regulates gene transcription and participates in DNA damage repair mechanisms . This dual localization makes ARPC2 a multifunctional protein involved in both structural and genetic regulatory processes within cells.

What are the most suitable applications for ARPC2 antibodies in research?

ARPC2 antibodies have been validated for multiple research applications with varying degrees of reliability. Western blotting (WB) represents the most widely used and consistently successful application, providing clear detection of the 34.3 kDa ARPC2 protein . Immunohistochemistry on paraffin-embedded tissues (IHC-P), immunocytochemistry/immunofluorescence (ICC/IF), and flow cytometry (intracellular) also demonstrate strong reliability for ARPC2 detection across human, mouse, and rat samples . Additional applications include immunoprecipitation (IP) for protein-protein interaction studies and enzyme-linked immunosorbent assay (ELISA) for quantitative analysis . When selecting an application, researchers should prioritize those that have been experimentally validated for their specific species of interest, as cross-reactivity varies between antibody clones.

What are the key considerations when selecting an ARPC2 antibody for my research?

When selecting an ARPC2 antibody, researchers should consider several critical factors. First, evaluate the antibody's validation status for your specific application and species (human, mouse, rat, etc.), as performance can vary significantly between experimental contexts . Second, consider the antibody format—monoclonal antibodies like EPR8533 offer high specificity and reproducibility, while polyclonal options may provide broader epitope recognition . Third, examine the immunogen sequence to ensure it will detect your protein of interest, particularly if studying specific isoforms. Fourth, review published literature citing the antibody to assess its reliability in similar experimental contexts. Finally, consider whether you need a conjugated antibody (e.g., fluorescent or enzyme-linked) or an unconjugated version suitable for custom labeling, depending on your downstream applications and detection systems .

What is the optimal protocol for immunofluorescence detection of ARPC2 in cultured cells?

For optimal immunofluorescence detection of ARPC2 in cultured cells, follow this validated protocol:

• Grow cells on sterile coverslips until 70-80% confluent.

• Fix cells with 4% paraformaldehyde for 15 minutes at room temperature.

• Permeabilize with 0.1% Triton X-100 in PBS for 10 minutes.

• Block with 5% normal serum (from the same species as the secondary antibody) in PBS with 0.1% Tween-20 for 1 hour.

• Incubate with primary ARPC2 antibody (e.g., EPR8533) at 1:100-1:250 dilution overnight at 4°C .

• Wash three times with PBS-T (PBS with 0.1% Tween-20).

• Incubate with fluorescently-labeled secondary antibody at 1:500 dilution for 1 hour at room temperature in the dark.

• Counterstain nuclei with DAPI (1:1000) for 5 minutes.

• Mount with anti-fade mounting medium.

This protocol reveals ARPC2's dual localization pattern in both cytoplasm and nucleus, with particular enrichment at areas of active actin polymerization in the cytoplasm. When co-staining with actin markers, ARPC2 shows notable co-localization at the leading edge of migrating cells and in lamellipodia, consistent with its role in branched actin network formation .

How can I effectively use ARPC2 antibodies for protein expression analysis in cancer tissues?

For effective analysis of ARPC2 protein expression in cancer tissues, implement this comprehensive approach:

• Tissue preparation: Use freshly fixed, paraffin-embedded tissue sections (4-6 μm thick) for IHC-P analysis. Include both tumor and adjacent normal tissues as comparisons .

• Antigen retrieval: Perform heat-mediated antigen retrieval using citrate buffer (pH 6.0) for 20 minutes, as this has been optimized for ARPC2 antibodies .

• Blocking and antibody incubation: Block with 10% normal serum and incubate with anti-ARPC2 antibody (1:100-1:200 dilution) overnight at 4°C .

• Detection system: Use a sensitive detection system such as HRP-polymer with DAB substrate for visualization.

• Scoring methodology: Implement a semi-quantitative scoring system that accounts for both staining intensity (0-3) and percentage of positive cells (0-100%), with a combined score calculated as: intensity × percentage of positive cells .

• Controls and validation: Include positive controls (tissues known to express ARPC2) and negative controls (primary antibody omitted). Additionally, validate IHC findings with Western blot analysis from the same tissue samples .

This approach has successfully demonstrated ARPC2 upregulation across multiple cancer types, with significantly higher expression in tumor tissues compared to adjacent normal tissues, correlating with clinicopathological parameters .

What are the recommended methods for silencing ARPC2 expression in functional studies?

For effective silencing of ARPC2 expression in functional studies, researchers should employ a multi-faceted approach:

• siRNA transfection: Design at least three different siRNA sequences targeting different regions of ARPC2 mRNA. Transfect cells using optimized reagents like TransIntro™ EL Transfection Reagent according to the manufacturer's protocol. Typical working concentrations range from 20-50 nM, with transfection efficiency varying by cell type .

• shRNA stable knockdown: For long-term studies, develop stable ARPC2-knockdown cell lines using lentiviral vectors carrying shRNA sequences. This approach provides more consistent and prolonged silencing compared to transient siRNA transfection.

• CRISPR-Cas9 gene editing: For complete knockout studies, design guide RNAs targeting exons of ARPC2 gene and introduce frameshifting indels using CRISPR-Cas9 technology.

• Validation of knockdown efficiency: Regardless of the silencing method, validate knockdown efficiency at both mRNA level (using qRT-PCR with primers targeting ARPC2) and protein level (using Western blot with anti-ARPC2 antibody) .

• Functional assays: After confirming ARPC2 knockdown, perform functional assays including proliferation (MTT/CCK-8), migration (wound healing/transwell), invasion (Matrigel-coated transwell), and actin cytoskeleton visualization (phalloidin staining) to assess phenotypic changes .

When performing these studies, always include appropriate controls (scrambled siRNA/shRNA or non-targeting gRNA) and test multiple cell lines to ensure reproducibility of observed phenotypes.

How does ARPC2 expression correlate with clinical outcomes in different cancer types?

ARPC2 expression demonstrates significant correlations with clinical outcomes across multiple cancer types, with particularly strong associations in epithelial cancers. Comprehensive pan-cancer analysis using TCGA and GTEx databases has revealed that ARPC2 is significantly upregulated in numerous malignancies compared to corresponding normal tissues . Higher ARPC2 expression consistently correlates with:

This consistent pattern across diverse cancer types suggests ARPC2 functions as a pan-cancer biomarker with prognostic significance. The mechanistic basis appears linked to ARPC2's fundamental role in actin cytoskeleton remodeling, which facilitates tumor cell motility and invasiveness, key processes in cancer progression and metastasis .

What is the relationship between ARPC2 expression and tumor immune microenvironment?

ARPC2 expression demonstrates significant correlations with tumor immune microenvironment parameters across various cancer types. Spearman correlation analyses between ARPC2 expression and immune-related metrics have revealed several consistent patterns:

• Immune cell infiltration: ARPC2 expression correlates with altered infiltration of specific immune cell populations. Higher ARPC2 levels are associated with decreased infiltration of CD8+ T cells and increased myeloid-derived suppressor cells in several cancer types, suggesting a potential immunosuppressive effect .

• Immune checkpoint markers: Significant positive correlations exist between ARPC2 expression and immune checkpoint genes (PD-1, PD-L1, CTLA4) in specific cancers, suggesting potential implications for immunotherapy response prediction .

• Tumor microenvironment scores: ESTIMATE algorithm analysis shows that ARPC2 expression correlates with stromal scores and immune scores in certain cancer types, indicating its relationship with the composition of the tumor microenvironment .

• Tumor mutational burden (TMB) and microsatellite instability (MSI): ARPC2 expression shows significant correlations with TMB and MSI in specific cancers, potentially influencing genomic instability and neoantigen presentation .

These findings suggest that ARPC2 may function not only as a driver of tumor cell intrinsic properties but also as a modulator of tumor-immune interactions, with potential implications for immunotherapy strategies and patient stratification.

What functional assays can best demonstrate ARPC2's role in cancer progression?

To comprehensively evaluate ARPC2's role in cancer progression, researchers should implement a multi-faceted functional assay approach:

• Proliferation assays:

  • MTT/CCK-8 colorimetric assays measuring cell viability over 24-96 hours

  • Colony formation assays evaluating long-term proliferative capacity

  • EdU incorporation assays to assess DNA synthesis rates

• Migration assays:

  • Wound healing (scratch) assays measuring collective cell migration

  • Transwell migration assays for chemotactic single-cell migration

  • Time-lapse microscopy tracking individual cell movement patterns

• Invasion assays:

  • Matrigel-coated transwell assays evaluating invasive capacity

  • 3D spheroid invasion assays in extracellular matrix mimetics

• Cytoskeletal visualization:

  • Phalloidin staining for F-actin visualization

  • Live-cell imaging with fluorescently tagged actin to monitor dynamics

  • Immunofluorescence co-localization of ARPC2 with other cytoskeletal markers

• In vivo models:

  • Xenograft tumor growth models comparing ARPC2-modified versus control cells

  • Metastatic colonization assays via tail vein injection

  • Orthotopic implantation models to assess invasion in physiological environments

Experimental evidence using these assays has demonstrated that ARPC2 silencing significantly inhibits cancer cell proliferation, migration, and invasion, while its overexpression enhances these oncogenic properties . Particularly in hepatocellular carcinoma, ARPC2 knockdown results in a 40-60% reduction in migration and invasion capacity, highlighting its critical role in cancer progression mechanisms related to cell motility and cytoskeletal remodeling .

What are common causes of false negative results in ARPC2 Western blotting, and how can they be resolved?

False negative results in ARPC2 Western blotting can stem from several technical issues, each requiring specific troubleshooting approaches:

• Inadequate protein extraction: ARPC2 is present in both nuclear and cytoplasmic compartments, requiring comprehensive extraction protocols .

  • Solution: Use RIPA buffer supplemented with protease inhibitors and perform sonication to ensure complete extraction from all cellular compartments.

• Suboptimal antigen retrieval: The epitope may be masked by protein folding or fixation.

  • Solution: Include denaturing agents (SDS, DTT) in sample buffer and heat samples at 95°C for 5 minutes before loading.

• Insufficient antibody concentration: Many protocols underestimate required antibody amounts.

  • Solution: Perform a titration experiment with 1:500, 1:1000, and 1:2000 dilutions to determine optimal concentration for your specific sample type .

• Inadequate transfer efficiency: ARPC2 (34.3 kDa) may transfer differently than other proteins.

  • Solution: Use Ponceau S staining to verify transfer efficiency and adjust transfer conditions (time, voltage) accordingly.

• Antibody specificity issues: Some antibodies may not recognize certain species or isoforms.

  • Solution: Select antibodies validated for your species and confirm recognition of human ARPC2 at 34.3 kDa band position .

• Sample degradation: ARPC2 protein may degrade during extended storage.

  • Solution: Use freshly prepared samples or add additional protease inhibitors before storage and avoid repeated freeze-thaw cycles.

In all troubleshooting scenarios, include a positive control (cell line known to express ARPC2) to distinguish between technical issues and true biological absence of expression.

How can I optimize ARPC2 immunohistochemistry staining in different tissue types?

Optimizing ARPC2 immunohistochemistry across diverse tissue types requires tissue-specific protocol adjustments:

• Fixation parameters:

  • Epithelial tissues: Standard 10% neutral buffered formalin fixation for 24 hours is optimal .

  • Brain tissues: Extend fixation to 48 hours to ensure adequate penetration.

  • Lymphoid tissues: Reduce fixation to 12-18 hours to prevent excessive crosslinking.

• Antigen retrieval optimization:

  • Liver/kidney: Heat-mediated retrieval in citrate buffer (pH 6.0) for 20 minutes provides optimal results .

  • Lung/breast: EDTA buffer (pH 9.0) often yields superior staining with reduced background.

  • Neural tissues: Extended retrieval (30 minutes) may be necessary due to dense tissue composition.

• Blocking strategy:

  • High-background tissues (liver, kidney): Double blocking with 3% hydrogen peroxide (10 min) followed by 5% normal serum (1 hour).

  • Low-background tissues: Standard single blocking with 3% normal serum is sufficient.

• Antibody dilution and incubation:

  • Tissues with high ARPC2 expression (tumors): Use 1:200-1:400 dilution to prevent overstaining .

  • Tissues with low expression: Use 1:50-1:100 dilution with overnight incubation at 4°C.

• Detection system selection:

  • Tissues with abundant target: Standard HRP-polymer systems are sufficient.

  • Tissues with sparse target: Amplification systems (e.g., tyramide signal amplification) may be necessary.

• Counterstaining adjustment:

  • Nuclear ARPC2 assessment: Light hematoxylin counterstaining (1-2 minutes).

  • Cytoplasmic ARPC2 assessment: Standard hematoxylin counterstaining (3-5 minutes).

These tissue-specific optimizations have been validated across multiple studies examining ARPC2 expression in normal and pathological samples .

What controls should be included when validating a new ARPC2 antibody for research applications?

Comprehensive validation of a new ARPC2 antibody requires a systematic approach incorporating multiple controls:

• Positive control tissues/cells:

  • Cell lines with confirmed ARPC2 expression (e.g., HepG2, HeLa)

  • Tissues with known high ARPC2 expression (e.g., liver, placenta)

  • Recombinant ARPC2 protein as positive control for Western blot

• Negative control tissues/cells:

  • ARPC2 knockout cell lines generated via CRISPR-Cas9

  • ARPC2 siRNA-treated cells showing knockdown verification

  • Primary antibody omission control for each application

• Specificity controls:

  • Peptide competition assay using the immunogen peptide

  • Western blot showing single band at expected molecular weight (34.3 kDa)

  • Immunoprecipitation followed by mass spectrometry verification

• Cross-reactivity assessment:

  • Testing across multiple species (human, mouse, rat) if cross-reactivity is claimed

  • Comparison with other validated ARPC2 antibodies targeting different epitopes

• Reproducibility controls:

  • Inter-lot consistency testing using multiple antibody lots

  • Inter-laboratory validation if possible

  • Consistent results across multiple experimental repeats

• Application-specific controls:

  • For IHC/ICC: Multiple fixation conditions

  • For flow cytometry: Isotype control antibodies

  • For IP: Non-immune IgG control

Documentation of all validation results should be maintained, including images of Western blots showing expected band size, immunostaining patterns consistent with known ARPC2 localization (nuclear and cytoplasmic), and correlation between detection methods (e.g., protein vs. mRNA levels).

How does nuclear ARPC2 contribute to DNA damage repair and gene expression regulation?

Nuclear ARPC2, as part of the Arp2/3 complex, plays critical roles in DNA damage repair and gene expression regulation through several sophisticated mechanisms:

• Homologous recombination (HR) repair facilitation:

  • Nuclear ARPC2 promotes homologous recombination repair of double-strand breaks (DSBs) by facilitating nuclear actin polymerization .

  • This polymerization generates mechanical forces that drive the mobility of DNA damage sites, increasing the probability of finding homologous templates for repair .

  • The process involves recruitment of the Arp2/3 complex to DSB sites through interactions with repair proteins like BRCA1 and CtIP.

• Chromatin remodeling dynamics:

  • ARPC2-mediated actin polymerization contributes to ATP-dependent chromatin remodeling.

  • This facilitates access of repair proteins to damaged DNA and transcription factors to gene regulatory elements.

  • The nuclear actin structures generated through ARPC2 activity serve as scaffolds for assembly of transcriptional complexes.

• Transcriptional regulation:

  • Nuclear ARPC2 associates with RNA polymerase II and influences transcriptional elongation rates.

  • It contributes to the formation of nuclear actin networks that regulate the spatial organization of transcriptionally active regions.

  • ARPC2-dependent actin polymerization affects chromatin looping and enhancer-promoter interactions.

• DNA damage response signaling:

  • ARPC2 participates in the activation of DNA damage checkpoints through interactions with signaling kinases.

  • Its activity influences cell cycle progression following DNA damage.

These nuclear functions represent an emerging area of research that expands ARPC2's known roles beyond cytoplasmic actin regulation. The dual functionality in both cytoplasmic and nuclear compartments positions ARPC2 as a critical coordinator of cellular responses to various stimuli, including genotoxic stress .

What are the key methodological considerations when studying ARPC2 interactions with other Arp2/3 complex components?

Studying ARPC2 interactions with other Arp2/3 complex components requires sophisticated methodological approaches that address the dynamic nature of these protein assemblies:

• Co-immunoprecipitation optimization:

  • Use mild detergent conditions (0.1% NP-40 or 0.5% CHAPS) to preserve native protein interactions .

  • Include crosslinking agents (DSP or formaldehyde at 0.1-1%) to capture transient interactions.

  • Perform reciprocal IPs using antibodies against different complex components (ARPC1, ARPC3, ARP2, ARP3) to validate interactions.

• Proximity-based interaction assays:

  • Implement BioID or TurboID proximity labeling by fusing biotin ligase to ARPC2.

  • Use FRET or BRET approaches with fluorescently tagged Arp2/3 components to measure direct interactions in living cells.

  • Apply APEX2 proximity labeling for electron microscopy visualization of interaction sites.

• Structural biology approaches:

  • Utilize cryo-electron microscopy for visualization of assembled Arp2/3 complex architecture.

  • Implement hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map interaction interfaces.

  • Apply cross-linking mass spectrometry (XL-MS) to identify specific residues involved in ARPC2-partner interactions.

• Functional interaction mapping:

  • Generate domain deletion and point mutation constructs of ARPC2 to map critical interaction regions.

  • Use competition assays with purified protein domains to disrupt specific interactions.

  • Implement mammalian two-hybrid or split-luciferase assays for quantitative interaction measurements.

• Dynamic interaction analysis:

  • Apply fluorescence recovery after photobleaching (FRAP) to measure exchange rates of ARPC2 within the complex.

  • Use single-molecule tracking to visualize real-time assembly/disassembly dynamics.

  • Implement optogenetic approaches to trigger on-demand complex formation or dissociation.

These methodological considerations help overcome challenges inherent in studying multi-component protein complexes like Arp2/3, where interactions may be cooperative, sequential, or context-dependent based on cellular activation state and localization .

How can ARPC2 expression patterns be leveraged for developing targeted cancer therapies?

The distinctive expression patterns and functional roles of ARPC2 in cancer present several promising avenues for developing targeted therapeutic strategies:

• Direct ARPC2 inhibition approaches:

  • Design of small molecule inhibitors targeting ARPC2's interaction with other Arp2/3 components.

  • Development of peptide mimetics that disrupt ARPC2's binding to actin filaments.

  • Exploration of RNA interference therapies (siRNA/shRNA) delivered via nanoparticle systems for ARPC2 silencing in tumors .

• ARPC2-based patient stratification:

  • Implementation of ARPC2 expression analysis as a companion diagnostic for treatment selection.

  • Development of immunohistochemistry scoring systems for ARPC2 to predict therapeutic responses.

  • Integration of ARPC2 with other biomarkers to create predictive signatures for treatment outcomes .

• Combination therapy approaches:

  • Targeting ARPC2 in conjunction with immune checkpoint inhibitors, as ARPC2 expression correlates with immune checkpoint markers in several cancers.

  • Combining cytoskeletal disruption via ARPC2 inhibition with conventional chemotherapies to enhance drug delivery and efficacy.

  • Dual targeting of ARPC2 and downstream effectors in migration/invasion pathways .

• Exploiting synthetic lethality:

  • Identification of genes synthetically lethal with high ARPC2 expression.

  • Development of combination approaches targeting both ARPC2 and DNA repair pathways, given ARPC2's role in homologous recombination.

  • Exploring metabolic vulnerabilities created by ARPC2 overexpression .

• Targeting ARPC2 in resistance mechanisms:

  • Addressing ARPC2-mediated migration/invasion as a mechanism of therapy resistance.

  • Development of maintenance therapies targeting ARPC2 to prevent metastatic progression after primary treatment.

  • Investigation of ARPC2's role in therapy-induced phenotypic transitions like epithelial-mesenchymal transition .