ARPC3 Antibody

What is ARPC3 and what role does it play in cellular function?

ARPC3, also known as p21-ARC or ARC21, is a 21 kDa subunit of the actin-related protein 2/3 complex (Arp2/3 complex). This complex plays a critical role in the nucleation of actin polymerization upon stimulation by nucleation-promoting factors (NPFs) . The Arp2/3 complex mediates the formation of branched actin networks in the cytoplasm, providing the force for cell motility .

Beyond its cytoplasmic function, the Arp2/3 complex also promotes actin polymerization in the nucleus, thereby regulating gene transcription and repair of damaged DNA . Specifically, it promotes homologous recombination (HR) repair in response to DNA damage by driving the motility of double-strand breaks (DSBs) . As part of the Arp2/3 complex, ARPC3 is essential for coordinating these diverse cellular processes involving actin dynamics.

What applications are ARPC3 antibodies validated for?

ARPC3 antibodies have been validated for multiple experimental applications, as shown in the following table:

It's important to note that individual antibodies may vary in their specific reactivity and optimal conditions. For example, antibody 14652-1-AP has been validated for WB, IHC, IF, IP, and ELISA applications with reactivity to human, mouse, and rat samples .

How should ARPC3 antibodies be stored and handled for optimal performance?

Proper storage and handling are critical for maintaining antibody activity and specificity. For ARPC3 antibodies:

• Store at -20°C for long-term storage. Antibodies are typically stable for one year after shipment when stored properly .

• For short-term storage and frequent use, some ARPC3 antibodies can be stored at 4°C for up to one month .

• Avoid repeated freeze-thaw cycles as they can degrade antibody quality and affect performance in experiments .

• Most ARPC3 antibodies are provided in storage buffers containing PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 .

• For small volumes (20 μl), aliquoting is generally unnecessary for -20°C storage .

• Some preparations may contain 0.1% BSA for stability .

When removing from storage, allow the antibody to equilibrate to room temperature before opening the vial to prevent condensation, which can affect antibody concentration and stability.

What positive controls are recommended for validating ARPC3 antibody experiments?

Selecting appropriate positive controls is essential for validating ARPC3 antibody specificity and experimental protocols:

Cell Lines:

• HeLa cells, Jurkat cells, K-562 cells, and NIH/3T3 cells have been validated as reliable positive controls for Western blot applications .

• U-251MG and SK cell lines are also reported as positive samples for ARPC3 detection .

Tissue Samples:

• For immunohistochemistry, human colon cancer tissue and mouse spleen tissue have been validated as positive controls .

• Mouse and rat brain samples have also been confirmed to express detectable levels of ARPC3 .

Protein Standards:

• Recombinant ARPC3 protein, particularly that corresponding to amino acids 1-178 of human ARPC3 (NP_001265485.1), can serve as a definitive positive control for antibody validation .

When validating a new ARPC3 antibody or experimental protocol, it is advisable to include at least one well-established positive control to ensure reliable results.

What is the recommended troubleshooting approach for weak or absent ARPC3 signals?

When faced with weak or absent signals in ARPC3 detection experiments, consider the following methodological troubleshooting steps:

For Western Blot:

• Adjust antibody concentration: Try a range of dilutions from 1:500 to 1:5000 . Some samples may require higher antibody concentrations for optimal detection.

• Increase protein loading: Since ARPC3 is a relatively small protein (21 kDa), higher protein loading may be necessary for clear detection.

• Optimize exposure time: Extending exposure time during imaging may capture weaker signals.

• Check transfer efficiency: Verify protein transfer using Ponceau S staining of the membrane.

• Verify sample integrity: Ensure samples are not degraded by adding fresh protease inhibitors during preparation.

For Immunohistochemistry/Immunofluorescence:

• Optimize antigen retrieval: The recommended method is TE buffer at pH 9.0, with citrate buffer at pH 6.0 as an alternative .

• Increase antibody concentration: Use dilutions at the lower end of the recommended range (1:50-1:100 for IF/ICC) .

• Extend incubation time: Overnight incubation at 4°C often improves signal strength.

• Test different fixation methods: Variations in fixation can significantly affect epitope accessibility.

General Considerations:

• Check antibody viability: Antibodies may lose activity after multiple freeze-thaw cycles or extended storage.

• Verify species reactivity: Ensure the antibody is validated for your experimental species .

• Consider blocking optimization: Test different blocking agents (BSA vs. milk) and concentrations.

• Secondary antibody compatibility: Confirm that secondary antibodies match the host species of your primary antibody.

How can ARPC3 antibodies be used to study the relationship between Arp2/3 complex and its inhibitors?

The interaction between the Arp2/3 complex and its inhibitors, such as Arpin, represents an important regulatory mechanism for actin dynamics. ARPC3 antibodies can be instrumental in studying these relationships:

Structural Analysis Approaches:

Recent cryo-EM studies have revealed that Arpin specifically binds to Arp3 rather than to other subunits of the complex . The high-resolution structure (3.24-Å) of Arpin bound to the Arp2/3 complex shows that Arpin's C-helix interacts with the C-terminal inhibitory tail of Arp3, effectively locking the complex in an inactive conformation .

To investigate these interactions:

• Co-immunoprecipitation studies: Use ARPC3 antibodies to pull down the Arp2/3 complex and analyze the co-precipitation of inhibitors under various cellular conditions.

• Competition assays: The structural data suggests that Arpin competes with nucleation-promoting factors (NPFs) specifically for binding to Arp3 . Design experiments to assess how varying concentrations of inhibitors affect NPF binding to the Arp2/3 complex.

• Mutational analysis: Based on structural insights, create point mutations in ARPC3 that might affect the binding of inhibitors without disrupting complex assembly. Use antibodies to assess complex formation and function in cells expressing these mutants.

• In vitro reconstitution: Combine purified components with ARPC3 antibodies to track complex assembly and inhibition in a controlled system.

What methods can be used to investigate ARPC3's role in nuclear actin polymerization and DNA repair?

The Arp2/3 complex has been implicated in nuclear actin polymerization, which regulates gene transcription and DNA repair processes . ARPC3 antibodies can be utilized to explore these nuclear functions:

Experimental Approaches:

• Subcellular fractionation and immunoblotting: Use ARPC3 antibodies (diluted 1:1000-1:5000) in Western blots of nuclear extracts to quantify nuclear localization of ARPC3 under different conditions .

• Chromatin immunoprecipitation (ChIP): Apply ARPC3 antibodies to identify genomic regions where the Arp2/3 complex associates with chromatin, particularly at sites of DNA damage.

• DNA damage response analysis:

  • Induce DNA damage using ionizing radiation or chemical agents

  • Use immunofluorescence with ARPC3 antibodies (1:50-1:100 dilution) to track recruitment to damage sites

  • Co-stain with markers of DNA double-strand breaks (γH2AX, 53BP1)

  • Quantify repair kinetics in cells with normal versus depleted ARPC3 levels

• Live-cell imaging: Generate cell lines expressing fluorescently tagged ARPC3 and validate expression using ARPC3 antibodies. Use these cells to monitor the real-time dynamics of ARPC3 during DNA damage and repair.

• Homologous recombination assays: Since the Arp2/3 complex specifically promotes homologous recombination repair , use reporter assays to measure HR efficiency in cells with manipulated ARPC3 levels.

How can super-resolution microscopy be combined with ARPC3 antibodies to study actin network architecture?

Super-resolution microscopy overcomes the diffraction limit of conventional microscopy, allowing visualization of actin networks at nanoscale resolution. Combining these techniques with ARPC3 antibodies enables detailed analysis of Arp2/3 complex localization and function:

Methodological Framework:

• Sample preparation optimization:

  • Fixation: Test different fixation protocols to preserve actin network architecture

  • Permeabilization: Optimize conditions to maintain network integrity while allowing antibody access

  • Antibody dilution: Start with 1:50-1:100 dilutions for immunofluorescence applications

• Multi-color imaging strategy:

  • ARPC3 labeling: Primary detection using validated ARPC3 antibodies

  • Actin visualization: Combine with phalloidin or actin antibodies to visualize filaments

  • Regulatory proteins: Include additional staining for NPFs or inhibitors like Arpin

• Quantitative analysis parameters:

  • Branching density: Measure the number of ARPC3-positive branches per unit length of actin filament

  • Branch angles: Quantify the angles between mother and daughter filaments

  • Spatial distribution: Analyze the clustering patterns of ARPC3 in different cellular regions

  • Co-localization metrics: Calculate precise spatial relationships between ARPC3 and other proteins

• Experimental manipulation contexts:

  • Drug treatments: Analyze network changes after treatment with actin or Arp2/3 complex inhibitors

  • Genetic manipulation: Compare networks in cells with normal versus altered ARPC3 expression

  • Stimulation responses: Track reorganization after growth factor or chemotactic stimulation

These super-resolution approaches enable researchers to connect molecular-level understanding of ARPC3 function with cellular-level actin network architecture and dynamics.

What considerations are important when using ARPC3 antibodies to study phosphorylation-dependent regulation of the Arp2/3 complex?

Post-translational modifications, particularly phosphorylation, represent an important regulatory mechanism for Arp2/3 complex function. When investigating these processes with ARPC3 antibodies, consider the following methodological approaches:

Experimental Design Considerations:

• Phospho-specific detection:

  • Standard ARPC3 antibodies may not distinguish between phosphorylated and non-phosphorylated forms

  • Consider using phospho-specific antibodies if available, or develop them for key regulatory sites

  • For Western blot applications, incorporate phosphatase inhibitors in sample preparation buffers

• Sample preparation protocols:

  • Cell lysis buffers should contain both protease inhibitors and phosphatase inhibitors

  • For optimal retention of phosphorylation, samples should be processed rapidly and kept cold

  • Consider sample fractionation to enrich for specific cellular compartments where phosphorylation may occur

• Functional correlation studies:

  • Design experiments that correlate ARPC3 phosphorylation status with functional outcomes like actin polymerization or cell migration

  • Use pharmacological inhibitors of kinases/phosphatases to modulate phosphorylation status

  • Express phosphomimetic (e.g., Ser→Glu) or phosphodeficient (e.g., Ser→Ala) ARPC3 mutants and verify expression using standard ARPC3 antibodies

• Temporal dynamics analysis:

  • Track changes in phosphorylation in response to stimuli that activate actin remodeling

  • Design time-course experiments with appropriate controls

  • Consider how phosphorylation timing relates to complex assembly and activation

How can ARPC3 antibodies be utilized in developing therapeutic approaches targeting cytoskeletal dynamics?

The Arp2/3 complex represents a potential therapeutic target for conditions involving dysregulated actin dynamics, such as cancer metastasis and certain inflammatory processes. ARPC3 antibodies can contribute to therapeutic research in several ways:

Research Applications for Therapeutic Development:

• Target validation studies:

  • Use ARPC3 antibodies in immunohistochemistry (1:500-1:2000 dilution) to assess expression patterns in disease tissues compared to normal tissues

  • Correlate expression levels with disease progression or clinical outcomes

  • Validate knockdown efficiency of therapeutic candidates using Western blot with ARPC3 antibodies (1:1000-1:5000 dilution)

• High-content screening applications:

  • Develop immunofluorescence-based assays (using ARPC3 antibodies at 1:50-1:100 dilution) to screen for compounds that affect Arp2/3 complex localization or function

  • Quantify changes in branched actin networks in response to candidate therapeutic agents

  • Measure effects on cellular processes dependent on Arp2/3 function, such as migration or invasion

• Mechanism of action studies:

  • For identified therapeutic candidates, use ARPC3 antibodies to elucidate whether they directly affect complex assembly, localization, or interaction with regulatory proteins

  • Combine with structural biology approaches to understand how therapeutics might alter complex conformation or activity

• Biomarker development:

  • Evaluate whether ARPC3 expression or specific post-translational modifications could serve as biomarkers for disease progression or treatment response

  • Develop standardized protocols for clinical sample analysis using validated ARPC3 antibodies