ARPC2B Antibody

What is ARPC2 and what is its function in cellular processes?

ARPC2 (actin-related protein 2/3 complex subunit 2) is a critical component of the Arp2/3 complex, a multiprotein assembly that mediates actin polymerization in response to stimulation by nucleation-promoting factors (NPFs). In humans, the canonical ARPC2 protein consists of 300 amino acid residues with a molecular mass of approximately 34.3 kDa . Its primary function involves binding to actin filaments and participating in the regulation of actin cytoskeleton dynamics. ARPC2 is widely expressed in various tissue types and plays fundamental roles in cellular processes requiring cytoskeletal remodeling, including cell migration, endocytosis, vesicle trafficking, and establishment of cell morphology. The protein's subcellular localization spans both nuclear and cytoplasmic compartments, suggesting diverse functional roles depending on cellular context .

What are the common applications for ARPC2 antibodies in research?

ARPC2 antibodies serve multiple research applications across cellular and molecular biology disciplines. Western blotting represents the most frequently utilized technique for detecting and quantifying ARPC2 protein expression in cell or tissue lysates . Immunohistochemistry (IHC) and immunocytochemistry (ICC) applications enable visualization of ARPC2 distribution patterns in tissue sections and cultured cells, respectively, providing spatial information about protein localization . Immunofluorescence (IF) techniques offer enhanced resolution for subcellular localization studies, particularly valuable for co-localization experiments with other cytoskeletal components. Additionally, ARPC2 antibodies are employed in enzyme-linked immunosorbent assays (ELISA) for quantitative analysis, and select antibodies demonstrate utility in immunoprecipitation (IP) applications for studying protein-protein interactions within the Arp2/3 complex .

What are the recommended sample preparation protocols for detecting ARPC2 in Western blot applications?

For optimal detection of ARPC2 in Western blot applications, sample preparation should begin with efficient cell lysis using buffers containing appropriate detergents (typically RIPA or NP-40-based buffers) and protease inhibitor cocktails to prevent protein degradation. Given ARPC2's molecular weight of 34.3 kDa, standard SDS-PAGE protocols using 10-12% polyacrylamide gels provide adequate resolution . Proteins should be transferred to PVDF or nitrocellulose membranes using semi-dry or wet transfer systems (25V for 1.5 hours in wet transfer systems is often effective). For immunodetection, membranes should be blocked with 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature. Primary ARPC2 antibody dilutions typically range from 1:500 to 1:2000 depending on the specific antibody , with overnight incubation at 4°C yielding optimal results. Following thorough washing with TBST, appropriate HRP-conjugated secondary antibodies should be applied at 1:5000-1:10000 dilutions for 1 hour at room temperature. After final washing steps, standard chemiluminescence detection methods can visualize ARPC2, which typically appears as a distinct band at approximately 34 kDa.

How should researchers optimize immunohistochemistry protocols for ARPC2 detection in tissue sections?

Optimizing immunohistochemistry protocols for ARPC2 detection requires careful consideration of fixation, antigen retrieval, and detection methods. Formalin-fixed paraffin-embedded (FFPE) tissues typically require heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) for 15-20 minutes . For frozen sections, acetone or paraformaldehyde fixation protocols are generally compatible with ARPC2 antibody detection. Blocking endogenous peroxidase activity with 3% hydrogen peroxide and non-specific binding with 5-10% normal serum from the species of the secondary antibody is recommended. Primary ARPC2 antibody incubation should be performed at dilutions ranging from 1:100 to 1:500, with overnight incubation at 4°C typically yielding superior staining compared to shorter incubations . Detection can utilize either chromogenic methods (DAB) with HRP-conjugated secondary antibodies or fluorescence-based visualization with fluorophore-conjugated secondary antibodies. ARPC2 exhibits both cytoplasmic and nuclear staining patterns in many cell types, with particularly strong expression in epithelial and immune cells. Validation should include appropriate positive control tissues with known ARPC2 expression and negative controls omitting primary antibody to confirm specificity.

What are the considerations for choosing between monoclonal and polyclonal ARPC2 antibodies?

The choice between monoclonal and polyclonal ARPC2 antibodies should be guided by specific experimental requirements and applications. Monoclonal antibodies offer superior specificity by recognizing a single epitope on the ARPC2 protein, making them ideal for applications requiring absolute specificity such as distinguishing between closely related proteins or detecting specific post-translational modifications . Their batch-to-batch consistency also benefits longitudinal studies. Several characterized monoclonal antibodies are available, including clone EPR8533, which has been validated for multiple applications including Western blot, immunohistochemistry, and immunofluorescence . Conversely, polyclonal antibodies recognize multiple epitopes on the ARPC2 protein, potentially offering enhanced sensitivity, particularly in applications where the target protein may be denatured or present at low concentrations. Many ARPC2 polyclonal antibodies target specific regions, such as the C-terminus or middle region, providing options for detecting different domains of the protein . Researchers should evaluate the validation data, including specificity testing against knockout samples, cross-reactivity profiles, and application-specific performance when selecting between these antibody types.

How can ARPC2 antibodies be utilized in studies of actin cytoskeleton dynamics and cell migration?

ARPC2 antibodies serve as powerful tools for investigating actin cytoskeleton dynamics and cell migration through multiple advanced approaches. In live-cell imaging studies, fluorescently tagged anti-ARPC2 antibody fragments can visualize the spatial and temporal recruitment of the Arp2/3 complex to sites of actin nucleation during lamellipodia formation and membrane protrusion. For fixed-cell analysis, dual immunofluorescence staining with ARPC2 antibodies combined with phalloidin (for F-actin) and antibodies against nucleation-promoting factors (e.g., WAVE complex components or WASP) can reveal the molecular organization of actin assembly machinery at leading edges during migration . In wound-healing assays or chemotaxis experiments, ARPC2 antibodies can track Arp2/3 complex redistribution as cells establish polarity and directional movement. Super-resolution microscopy techniques such as STORM or PALM, when combined with ARPC2 immunostaining, can provide nanoscale resolution of Arp2/3 complex organization within actin networks. Additionally, researchers can employ ARPC2 antibodies in proximity ligation assays (PLA) to investigate protein-protein interactions between ARPC2 and other cytoskeletal regulators in situ, offering insights into the molecular mechanisms controlling actin polymerization dynamics during migration and invasion processes.

What methodological approaches can be used to investigate ARPC2 interactions with other components of the Arp2/3 complex?

Investigating ARPC2 interactions with other Arp2/3 complex components requires sophisticated biochemical and imaging methodologies. Co-immunoprecipitation (Co-IP) using ARPC2 antibodies represents a fundamental approach, where cell lysates are incubated with antibodies to precipitate ARPC2 along with its interacting partners for subsequent analysis by Western blotting or mass spectrometry . For analyzing the dynamic assembly of the complex, researchers can employ fluorescence resonance energy transfer (FRET) by labeling ARPC2 antibodies and antibodies against other subunits (ARPC1, ARPC3-5, ARP2, ARP3) with appropriate donor-acceptor fluorophore pairs, enabling real-time monitoring of protein proximity. Proximity-dependent biotinylation (BioID or TurboID) approaches, where ARPC2 is fused to a biotin ligase, can identify transient or weak interactions within the complex in living cells. Cryo-electron microscopy using gold-labeled ARPC2 antibodies can visualize the structural organization of the Arp2/3 complex at molecular resolution. Additionally, chromatin immunoprecipitation (ChIP) using ARPC2 antibodies can investigate potential nuclear functions, as ARPC2 exhibits nuclear localization . For functional studies, researchers can deplete specific Arp2/3 components using siRNA or CRISPR-Cas9 approaches, followed by immunoprecipitation with ARPC2 antibodies to assess complex integrity and stability in the absence of individual subunits.

How can researchers troubleshoot non-specific binding issues when using ARPC2 antibodies?

Addressing non-specific binding with ARPC2 antibodies requires systematic troubleshooting across multiple parameters. First, researchers should verify antibody specificity through rigorous controls, including ARPC2 knockout or knockdown samples, which should demonstrate significant reduction or absence of the target band or signal . Optimization of blocking conditions is critical—testing alternative blocking agents such as 5% BSA, 5% normal serum, or commercial blocking reagents can reduce background, particularly in immunohistochemistry and immunofluorescence applications. Titration experiments across broader antibody dilution ranges (1:100 to 1:5000) can identify the optimal concentration that maximizes specific signal while minimizing background. For Western blotting applications, stringent washing protocols with increased detergent concentration (0.1-0.3% Tween-20) in wash buffers and extended washing times can significantly reduce non-specific binding. When persistent cross-reactivity occurs, pre-adsorption of the ARPC2 antibody with recombinant ARPC2 protein can confirm specificity—signals that disappear after pre-adsorption represent specific detection. For immunohistochemistry, antigen retrieval optimization is crucial, as insufficient retrieval can reduce specific binding while excessive retrieval may increase non-specific interactions. Finally, switching to alternative ARPC2 antibodies targeting different epitopes may resolve specificity issues, as some regions of the protein may share homology with other proteins, particularly other Arp2/3 complex components.

What role does ARPC2 play in cancer progression and metastasis, and how can antibodies facilitate this research?

ARPC2's involvement in cancer progression and metastasis represents an emerging research area where antibodies serve as essential investigative tools. ARPC2 participates in actin cytoskeleton remodeling that drives invasive phenotypes, with altered expression reported in multiple cancer types. Immunohistochemical analysis using ARPC2 antibodies on tissue microarrays can quantitatively assess expression patterns across tumor stages and correlate levels with clinical outcomes and metastatic potential . In migration and invasion assays, immunofluorescence with ARPC2 antibodies can visualize its recruitment to invadopodia structures, where localized actin polymerization facilitates extracellular matrix degradation. Researchers can employ ARPC2 antibodies in combination with phospho-specific antibodies to investigate its activation status in response to oncogenic signaling pathways. For mechanistic studies, chromatin immunoprecipitation sequencing (ChIP-seq) using ARPC2 antibodies can identify potential transcriptional regulatory functions in the nucleus, as ARPC2 shows nuclear localization . In animal models, antibody-based imaging techniques can track ARPC2-expressing tumor cells during metastatic dissemination. Additionally, proximity ligation assays using ARPC2 antibodies paired with antibodies against known cancer-promoting factors can reveal novel interaction networks specific to malignant contexts, potentially identifying therapeutic targets within the actin regulatory machinery involved in cancer progression.

How can ARPC2 antibodies be applied in neuroscience research to study cytoskeletal dynamics in neurons?

ARPC2 antibodies offer valuable approaches for investigating neuronal cytoskeletal dynamics crucial for development, plasticity, and pathology. In developmental neurobiology, immunohistochemistry and immunofluorescence with ARPC2 antibodies can track Arp2/3 complex localization during neurite outgrowth, growth cone navigation, and dendritic spine formation . High-resolution imaging of cultured neurons immunostained for ARPC2 can reveal its distribution within specialized structures such as dendritic spines, where actin remodeling underlies synaptic plasticity. Time-course studies utilizing ARPC2 antibodies can examine its dynamics during long-term potentiation or depression protocols. For investigating neurodegenerative conditions, immunohistochemical analysis of post-mortem brain tissues can assess ARPC2 distribution in relation to pathological hallmarks such as amyloid plaques or neurofibrillary tangles. In axonal injury models, ARPC2 antibodies can track cytoskeletal reorganization during regenerative responses. Live imaging of neurons using fluorescent nanobodies derived from ARPC2 antibodies permits real-time visualization of cytoskeletal dynamics. Additionally, researchers can employ ARPC2 antibodies in co-immunoprecipitation studies from brain tissue to identify neuron-specific interacting partners that may differ from other cell types. When combined with electrophysiological recordings, ARPC2 immunostaining can correlate cytoskeletal organization with functional synaptic properties, providing insights into structure-function relationships in neuronal connectivity.

What methodological considerations should be addressed when using ARPC2 antibodies in high-content screening applications?

Implementing ARPC2 antibodies in high-content screening (HCS) applications requires careful methodological considerations to ensure robust, reproducible results suitable for large-scale analysis. First, antibody validation through relevant positive and negative controls is essential, with ARPC2 knockdown or knockout samples serving as definitive specificity controls . Optimization of fixation and permeabilization protocols specifically for HCS plate formats is critical—paraformaldehyde fixation (4%, 15 minutes) followed by Triton X-100 permeabilization (0.1%, 10 minutes) typically yields consistent ARPC2 staining while preserving cellular architecture. Researchers should conduct preliminary Z'-factor analysis using positive and negative controls to assess assay quality and suitability for screening applications. Antibody concentration titration in the actual screening format is necessary, as optimal dilutions often differ from standard immunofluorescence protocols due to differences in surface-to-volume ratios in multiwell plates. For multiplexed approaches, careful selection of compatible antibodies raised in different host species allows simultaneous detection of ARPC2 alongside other markers. Automated image acquisition parameters require optimization for ARPC2 subcellular localization patterns, including appropriate exposure settings, Z-stack requirements, and objective magnification. Feature extraction algorithms should be specifically trained to recognize ARPC2 distribution patterns of interest, such as leading-edge localization or nuclear-to-cytoplasmic ratios. Finally, implementation of batch correction methods and inclusion of plate normalization controls mitigates technical variability across plates and experimental days, ensuring data comparability throughout large-scale screening campaigns.

How might ARPC2 antibodies contribute to understanding post-translational modifications regulating Arp2/3 complex function?

ARPC2 antibodies hold significant potential for elucidating the complex landscape of post-translational modifications (PTMs) that regulate Arp2/3 complex function. Development of modification-specific antibodies that recognize phosphorylated, acetylated, or ubiquitinated forms of ARPC2 would enable precise tracking of these regulatory events in various cellular contexts . Phospho-specific ARPC2 antibodies would be particularly valuable, as phosphorylation events on the Arp2/3 complex are known to modulate its nucleation activity. Researchers could employ these specialized antibodies in cell stimulation time-course experiments to correlate specific modifications with functional outcomes in actin polymerization. Mass spectrometry analysis following immunoprecipitation with pan-ARPC2 antibodies can identify novel modification sites that could subsequently become targets for new modification-specific antibody development. Proximity ligation assays combining ARPC2 antibodies with antibodies against modification-specific enzymes (kinases, phosphatases, acetyltransferases) could reveal the enzymatic machinery responsible for dynamically regulating ARPC2 status. Super-resolution microscopy using pairs of antibodies recognizing modified and unmodified ARPC2 forms could provide spatial information about modification patterns within cellular structures. Additionally, conformation-specific antibodies might detect structural changes in ARPC2 induced by modifications, offering insights into how PTMs alter complex assembly and activation states in response to different cellular signals.

What methodological advances might enhance the application of ARPC2 antibodies in live-cell imaging studies?

Advancing ARPC2 antibody applications for live-cell imaging requires innovative adaptations to overcome traditional limitations of antibody-based detection in living systems. Development of cell-permeable mini-antibodies or nanobodies derived from conventional ARPC2 antibodies represents a promising approach—these smaller antibody fragments can penetrate live cell membranes while maintaining specificity . Genetic engineering of fluorescent protein-tagged intrabodies (intracellular antibodies) specifically targeting ARPC2 would enable stable expression systems for long-term visualization of dynamic ARPC2 behavior. Antibody-based FRET biosensors could be designed to detect ARPC2 conformational changes or interactions with other Arp2/3 components in real-time. Site-specific labeling techniques that conjugate small, bright fluorophores to ARPC2 antibody fragments at precise locations would minimize functional interference while maximizing signal. Advanced delivery methods such as microinjection, electroporation, or lipid-based transfection specifically optimized for antibody delivery could improve intracellular access. Integration with emerging light-sheet microscopy technologies would enable longer imaging periods with reduced phototoxicity, critical for capturing ARPC2 dynamics during extended cellular processes such as differentiation or migration. Finally, combining ARPC2 antibody-based imaging with optogenetic approaches could allow simultaneous visualization and manipulation of Arp2/3 complex activity, providing unprecedented insights into cause-effect relationships in actin cytoskeleton regulation during dynamic cellular events.