ACTR2 Antibody

What is ACTR2 and what cellular functions does it regulate?

ACTR2, also known as ARP2 or actin-like protein 2, is a key component of the Arp2/3 complex that mediates actin polymerization following stimulation by nucleation-promoting factors (NPFs). In humans, the canonical protein consists of 394 amino acid residues with a molecular mass of approximately 44.8 kDa . The protein is localized in both the nucleus and cytoplasm, with up to two different reported isoforms .

ACTR2 plays several critical cellular roles. As part of the Arp2/3 complex, it mediates the formation of branched actin networks in the cytoplasm, providing the force necessary for cell motility . Within this complex, ACTR2 appears to contact the pointed end of the daughter actin filament . In podocytes, ACTR2 is required for the formation of lamellipodia downstream of AVIL and PLCE1 regulation .

Beyond its cytoplasmic functions, ACTR2 also promotes actin polymerization in the nucleus, thereby regulating gene transcription and DNA repair mechanisms . Specifically, the Arp2/3 complex promotes homologous recombination (HR) repair in response to DNA damage by facilitating the motility of double-strand breaks (DSBs) . This dual functionality in both cytoplasmic and nuclear compartments makes ACTR2 a multifaceted regulator of cellular architecture and genomic integrity.

ACTR2 is widely expressed across numerous tissue types, and orthologs have been identified in multiple species including mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken .

What types of ACTR2 antibodies are available and how should they be selected for specific applications?

Researchers have access to several types of ACTR2 antibodies that vary in source, specificity, and validated applications:

• Monoclonal antibodies:

  • Mouse monoclonal antibodies such as clone CD01/1A6 and FMS96 provide high specificity for human ACTR2.

  • These antibodies recognize specific epitopes and typically offer consistent results across experiments.

  • Monoclonal antibodies are particularly valuable for applications requiring high reproducibility.

• Polyclonal antibodies:

  • Rabbit polyclonal antibodies like 10922-1-AP recognize multiple epitopes of ACTR2 .

  • These antibodies often provide stronger signals due to binding at multiple sites but may exhibit batch-to-batch variation.

  • Polyclonal antibodies can be advantageous for detecting proteins present at low concentrations.

• Region-specific antibodies:

  • Options include middle region-specific antibodies (e.g., ARP46021_P050) and N-terminal region antibodies targeting amino acids 1-160 (e.g., STJ28301) .

  • The epitope location can affect detection in different experimental contexts, particularly if structural features mask certain regions.

When selecting an ACTR2 antibody, researchers should consider:

• Species reactivity: Most commercial ACTR2 antibodies react with human samples, while many cross-react with mouse and rat ACTR2 due to high sequence homology .

• Validated applications: Different antibodies are optimized for specific techniques. For example, 10922-1-AP is validated for Western blot, IHC, IF/ICC, and flow cytometry , while others may have more limited application profiles.

• Experimental requirements: Consider whether native conformation recognition is necessary (for IP or IF) versus denatured protein detection (for Western blot).

• Subcellular localization studies: Some antibodies may preferentially detect nuclear or cytoplasmic pools of ACTR2 depending on epitope accessibility in different cellular compartments.

What are the optimal protocols for using ACTR2 antibodies in common laboratory techniques?

ACTR2 antibodies are employed in various research techniques, each requiring specific optimization for reliable results:

• Western Blotting (WB):

  • ACTR2 typically appears as a band at approximately 43-45 kDa .

  • Recommended dilutions range from 1:1000-1:4000 for most antibodies .

  • Sample preparation should include phosphatase inhibitors as phosphorylation affects ACTR2 function .

  • Validated in multiple cell lines including HeLa, Jurkat, MCF-7, and SH-SY5Y cells .

  • BSA is often preferred over milk for blocking buffer as milk proteins may interact with some actin-binding proteins.

• Immunohistochemistry (IHC):

  • Optimal dilutions typically range from 1:200-1:800 .

  • Antigen retrieval is critical; TE buffer pH 9.0 is recommended, though citrate buffer pH 6.0 may also be effective .

  • Human prostate cancer tissue has been validated as a positive control .

  • Background reduction may require extended blocking steps (1-2 hours) with normal serum from the secondary antibody host species.

• Immunofluorescence (IF)/Immunocytochemistry (ICC):

  • Dilutions from 1:50-1:500 are typically effective .

  • Fixation method affects epitope preservation; compare paraformaldehyde (maintains structure) versus methanol (better for some epitopes).

  • Successfully used in cell lines including U-251 .

  • Co-staining with actin markers requires careful antibody selection to avoid epitope masking.

• Flow Cytometry:

  • For intracellular detection, use approximately 0.40 μg per 10^6 cells in a 100 μl suspension .

  • Effective permeabilization is essential; try saponin (0.1%) for gentler permeabilization compared to Triton X-100.

  • Include proper gating controls and isotype controls for accurate interpretation.

• Immunoprecipitation (IP):

  • Lysis buffer composition is critical; consider adding ATP (1-2 mM) to maintain ACTR2's native conformation.

  • The Arp2/3 complex stability is salt-sensitive; test buffers with varying ionic strengths.

  • Include appropriate controls: IgG matched to the host species of the ACTR2 antibody and input samples.

For all applications, optimization should include antibody titration, incubation time/temperature testing, and validation with positive and negative controls. Cell-type specific optimization may be necessary as ACTR2 expression and localization patterns vary across different tissues and cell types.

How can researchers optimize ACTR2 antibody performance in challenging tissue types?

Optimizing ACTR2 antibody performance in challenging tissue types requires a systematic approach addressing tissue-specific characteristics:

• Preliminary tissue assessment:

  • Before selecting an antibody, analyze ACTR2 expression levels in your target tissue using public databases.

  • While ACTR2 is widely expressed , expression levels vary significantly across tissues.

  • For tissues with known low expression, plan for signal amplification methods from the outset.

• Fixation and antigen retrieval optimization:

  • Conduct a fixation matrix experiment testing multiple fixatives (paraformaldehyde, methanol, acetone) and durations.

  • For ACTR2 IHC, compare heat-induced epitope retrieval using TE buffer pH 9.0 versus citrate buffer pH 6.0 .

  • Enzymatic retrieval methods may preserve morphology better in certain tissues but can affect some epitopes.

  • For highly autofluorescent tissues like brain or liver, consider using Sudan Black B (0.1-0.3%) post-staining to reduce background.

• Antibody selection strategies:

  • For fibrous tissues (muscle, connective tissue), monoclonal antibodies often provide better specificity .

  • For tissues with complex matrix proteins, polyclonal antibodies may offer superior sensitivity .

  • Region-specific antibodies may perform differently depending on protein interactions in specific tissues.

  • Consider using multiple antibodies recognizing distinct epitopes as internal validation.

• Signal amplification methods:

  • For tissues with low ACTR2 expression, implement:

    • Tyramide signal amplification (TSA), which can increase sensitivity 10-100 fold

    • Polymer-based detection systems that carry multiple secondary antibodies and enzymes

    • Extended primary antibody incubation (overnight at 4°C) with gentle agitation

• Background reduction for specific tissue challenges:

  • For tissues with high endogenous peroxidase (liver, kidney): Implement dual peroxidase blocking (3% H₂O₂ followed by peroxidase blocking reagent)

  • For tissues with high endogenous biotin (kidney, brain): Use avidin/biotin blocking kit before antibody application

  • For fatty tissues: Include additional blocking with 5% nonfat dry milk or commercial protein blockers

• Tissue-specific control strategies:

  • Always include positive control tissues with known ACTR2 expression patterns

  • Implement absorption controls by pre-incubating antibody with recombinant ACTR2 protein

  • For tissues with high background, include isotype controls at the same concentration as the primary antibody

By systematically addressing these factors, researchers can develop robust protocols for detecting ACTR2 across diverse and challenging tissue types while maintaining specificity and sensitivity.

What strategies can resolve non-specific binding issues with ACTR2 antibodies?

Non-specific binding presents a significant challenge when working with ACTR2 antibodies. A methodical troubleshooting approach includes:

• Diagnostics to identify true non-specific binding:

  • Multiple unexpected bands in Western blot beyond the expected 43-45 kDa

  • Diffuse rather than discrete cellular staining in IF/ICC

  • Persistent signal in negative control samples (e.g., ACTR2 knockdown cells)

  • Signal in tissues known to have minimal ACTR2 expression

• Antibody validation and selection refinement:

  • Test multiple antibodies targeting different epitopes of ACTR2

  • Compare results from monoclonal and polyclonal antibodies

  • Perform peptide competition assays to confirm specificity

  • Verify antibody specificity using ACTR2 knockdown/knockout samples

  • Consider the isoform specificity of each antibody, as ACTR2 has two reported isoforms

• Protocol modifications to enhance specificity:

  • Blocking optimization:

    • Extend blocking time (1-2 hours at room temperature or overnight at 4°C)

    • Test different blocking agents (BSA, normal serum from secondary antibody host, commercial blockers)

    • For problematic tissues, implement dual blocking with protein and detergent-based blockers

  • Antibody dilution adjustments:

    • For Western blot: Test higher dilutions (1:2000-1:4000)

    • For IHC: Begin with higher dilutions (1:500-1:800) and titrate as needed

    • For IF/ICC: Consider more dilute preparations (1:250-1:500)

  • Washing stringency:

    • Increase wash duration (5-10 minutes per wash)

    • Add additional wash steps (5-6 washes instead of 3)

    • Include higher detergent concentrations (0.1-0.2% Tween-20 or 0.05% Triton X-100)

• Application-specific solutions:

  • For Western blot:

    • Pre-adsorb antibodies against species-matched negative control lysates

    • Use gradient gels to improve resolution around 43-45 kDa

    • Consider membrane type (PVDF vs. nitrocellulose) as protein binding characteristics differ

  • For IHC/IF:

    • Implement tissue-specific permeabilization optimization

    • Test different secondary antibody formats (F(ab')2 fragments to reduce Fc receptor binding)

    • Use confocal microscopy to better distinguish specific subcellular localization

• Advanced strategies for persistent issues:

  • Antibody purification:

    • Affinity purify polyclonal antibodies against the immunogen

    • Deplete cross-reactive antibodies using negative selection with knockout tissue lysates

  • Signal verification with orthogonal methods:

    • Confirm ACTR2 localization with fluorescently tagged ACTR2 in transfected cells

    • Validate with alternative detection methods (e.g., mass spectrometry, RNA expression)

By systematically implementing these strategies, researchers can significantly improve specificity when working with ACTR2 antibodies across different experimental platforms and sample types.

How should researchers approach ACTR2 antibody use in co-immunoprecipitation and protein-protein interaction studies?

Co-immunoprecipitation (Co-IP) with ACTR2 antibodies presents unique challenges due to ACTR2's involvement in multi-protein complexes. Here's a comprehensive approach:

• Antibody selection for optimal Co-IP:

  • Choose antibodies specifically validated for immunoprecipitation applications

  • Consider using monoclonal antibodies for higher specificity when investigating specific interactions

  • Polyclonal antibodies may provide advantages for capturing the entire Arp2/3 complex

  • Confirm the antibody recognizes native (non-denatured) ACTR2

  • Test multiple antibodies targeting different epitopes to avoid interference with protein-protein interaction sites

• Cell lysis and sample preparation optimization:

  • Buffer composition is critical for preserving interactions:

    • Avoid harsh detergents like SDS or deoxycholate that disrupt protein-protein interactions

    • Start with mild NP-40 (0.5-1%) or digitonin (0.5-1%) based buffers

    • Include ATP (1-2 mM) to maintain ACTR2's native conformation as an ATP-binding protein

  • Preservation of post-translational modifications:

    • Add phosphatase inhibitors as phosphorylation of Arp2 by NIK is crucial for complex activity

    • Include protease inhibitor cocktail to prevent degradation during processing

  • Cell harvesting and lysis conditions:

    • Maintain samples at 4°C throughout processing

    • Consider crosslinking with formaldehyde (0.1-0.5%) or DSP for transient interactions

    • For nuclear ACTR2 interactions , compare cytoplasmic versus nuclear fraction preparations

• Immunoprecipitation procedure refinements:

  • Pre-clearing strategy:

    • Pre-clear lysates with protein A/G beads and control IgG to reduce non-specific binding

    • Use blocking agents (BSA, salmon sperm DNA) to minimize non-specific interactions

  • Antibody coupling methods:

    • Direct conjugation to beads can reduce background from heavy/light chains

    • Covalent coupling using crosslinkers for clean elution without antibody contamination

  • Washing stringency balance:

    • Implement graduated washing with increasing salt concentrations (150-300 mM)

    • Fine-tune detergent concentration to maintain specific interactions while reducing background

• Comprehensive controls for result validation:

  • Negative controls:

    • IgG matched to the host species of the ACTR2 antibody

    • ACTR2 knockdown/knockout samples to confirm specificity

  • Positive controls:

    • Immunoprecipitate known Arp2/3 complex components (e.g., Arp3)

    • Reciprocal IP with antibodies against established interaction partners

  • Specificity controls:

    • Peptide competition to block specific antibody binding

    • Input samples (5-10% of pre-cleared lysate) to confirm target protein presence

• Detection strategy for interaction partners:

  • Targeted Western blot analysis:

    • Probe for known Arp2/3 complex components

    • Investigate hypothesized interacting proteins with specific antibodies

  • Unbiased discovery approaches:

    • Mass spectrometry analysis of immunoprecipitates for novel interactors

    • Comparative proteomics between different cellular conditions

• Validation of identified interactions:

  • Orthogonal methodologies:

    • Proximity ligation assay (PLA) to confirm interactions in intact cells

    • FRET/BRET analysis for direct protein-protein interactions

    • Split reporter assays (e.g., split luciferase) for dynamic interaction studies

  • Functional validation:

    • Test the effect of actin-disrupting drugs on detected interactions

    • Evaluate interaction changes during cell stimulation or stress conditions

By following this systematic approach, researchers can effectively use ACTR2 antibodies to study protein-protein interactions while minimizing artifacts and increasing confidence in the biological relevance of results.

How should researchers account for ACTR2 subcellular localization when interpreting antibody signals?

ACTR2's dual localization in both cytoplasmic and nuclear compartments creates complexities for data interpretation that require methodological attention:

• Compartment-specific detection considerations:

  • ACTR2 localizes to both nucleus and cytoplasm , with distinct functions in each compartment

  • Nuclear ACTR2 participates in actin polymerization for gene transcription and DNA repair

  • Cytoplasmic ACTR2 functions within the Arp2/3 complex for cell motility and membrane dynamics

  • Antibody epitope accessibility may differ between compartments due to complex formation and protein interactions

• Subcellular fractionation approaches:

  • Implement differential centrifugation to separate nuclear and cytoplasmic fractions

  • Verify fraction purity using compartment-specific markers (e.g., GAPDH for cytoplasm, Lamin B1 for nucleus)

  • Quantify ACTR2 distribution across fractions using validated antibodies

  • Control for cross-contamination that could lead to misinterpretation of localization patterns

• Advanced imaging strategies:

  • Confocal microscopy with z-stack acquisition to precisely locate signals in 3D

  • Super-resolution microscopy to distinguish between adjacent structures beyond diffraction limit

  • Co-localization analysis with compartment-specific markers:

    • Nuclear markers: DAPI, Lamin B1, Histone H3

    • Cytoskeletal markers: Phalloidin (F-actin), α-tubulin

    • Membrane compartments: Markers for different cellular membranes if relevant

• Signal quantification methods:

  • Develop masks for different cellular compartments for accurate signal quantification

  • Implement intensity correlation analysis rather than simple overlay for co-localization

  • Consider pixel-by-pixel analysis using Pearson's or Mander's correlation coefficients

  • Account for background levels in each compartment separately

• Biological state considerations:

  • Cell cycle effects on ACTR2 localization:

    • Enhanced nuclear localization during S-phase for DNA repair functions

    • Dynamic redistribution during mitosis

  • Stimulus-induced relocalization:

    • Migration-inducing factors may recruit ACTR2 to the leading edge

    • DNA damage can increase nuclear accumulation

  • Cell confluence effects:

    • Contact inhibition alters cytoskeletal organization and may affect ACTR2 distribution

• Interpretation challenges and solutions:

  • Signal intensity variations:

    • Concentrated ACTR2 in small compartments may appear brighter than diffuse distribution

    • Normalize to compartment volume or area for accurate comparisons

  • Fixation artifacts:

    • Compare multiple fixation methods as they can artificially alter protein localization

    • Validate key findings with live-cell imaging of tagged ACTR2 when possible

  • Threshold setting:

    • Implement consistent and objective thresholding across samples

    • Consider automated algorithms to remove observer bias

What factors affect ACTR2 antibody performance in quantitative assays?

Multiple factors influence ACTR2 antibody performance in quantitative assays, requiring careful consideration for accurate results:

• Antibody characteristics affecting quantitation:

  • Affinity variation:

    • Different antibodies exhibit varying affinities for ACTR2, affecting signal intensity

    • Monoclonal antibodies typically provide more consistent affinity across experiments

    • Polyclonal antibodies may have batch-to-batch variation affecting quantitative comparisons

  • Epitope accessibility:

    • Antibodies targeting different regions of ACTR2 may have differential access depending on protein conformation

    • Middle-region versus N-terminal antibodies may yield different quantitative results

  • Linear detection range:

    • Each antibody has a specific range where signal correlates linearly with protein concentration

    • Determine this range through standard curve analysis before quantitative experiments

• Sample preparation factors:

  • Protein extraction efficiency:

    • Different lysis buffers extract ACTR2 with varying efficiency from subcellular compartments

    • Compare RIPA, NP-40, and urea-based buffers for total protein recovery

  • Post-translational modifications:

    • Phosphorylation of ACTR2 by NIK affects its activity and potentially antibody recognition

    • Sample handling can alter modification states (e.g., phosphatase activity during preparation)

  • Complex dissociation conditions:

    • ACTR2 exists in the Arp2/3 complex; harsh conditions may improve detection but disrupt native state

    • Gentle conditions preserve complexes but may mask epitopes

• Assay-specific considerations:

  • Western blot quantification:

    • Signal saturation at high protein concentrations creates non-linear response

    • Use gradient loading to determine linear detection range for each antibody

    • Consider fluorescent secondary antibodies for wider linear dynamic range

  • ELISA/quantitative IF:

    • Develop standard curves using recombinant ACTR2 protein

    • Account for matrix effects from cellular components

    • Implement plate layout strategies to control for edge effects

• Normalization strategies:

  • Loading control selection:

    • Traditional housekeeping proteins may vary across experimental conditions

    • Consider total protein normalization methods (Ponceau S, REVERT stain)

    • For subcellular fractions, use compartment-specific loading controls

  • Technical normalization:

    • Include internal reference standards on each gel/plate

    • Implement technical replicates to account for transfer/detection variation

    • Consider multiplexed detection systems for simultaneous target and control measurement

• Common pitfalls and solutions:

  • Non-specific background:

    • Implement robust blocking procedures appropriate for each application

    • Optimize antibody concentrations through titration experiments

    • Subtract local background for each measurement region

  • Signal variability between replicates:

    • Standardize all processing steps with precise timing

    • Use automated liquid handling where possible

    • Implement quality control metrics to identify outliers

• Advanced quantitative approaches:

  • Absolute quantification:

    • Develop calibrated standard curves using purified ACTR2 protein

    • Consider spike-in standards for mass spectrometry-based quantification

  • Single-cell quantification:

    • Account for cell-to-cell variability in ACTR2 expression

    • Develop normalization strategies for cell size and morphology

    • Consider flow cytometry for high-throughput single-cell analysis

By systematically addressing these factors, researchers can develop robust quantitative assays for ACTR2 that provide reliable and reproducible results across different experimental conditions.

How can contradictory results from different ACTR2 antibodies be reconciled?

Researchers frequently encounter contradictory results when using different ACTR2 antibodies. A systematic approach to reconciling these differences includes:

How are ACTR2 antibodies contributing to our understanding of disease mechanisms?

ACTR2 antibody research has revealed important connections between cytoskeletal regulation and disease pathogenesis:

• ACTR2 in cancer biology:

  • Diagnostic and prognostic insights in osteosarcoma:

    • Recent studies demonstrated significantly lower ACTR2 expression in osteosarcoma patients compared to healthy controls

    • Three-year prognostic follow-up revealed significantly lower survival rates among patients with low ACTR2 expression compared to those with high expression

    • ACTR2 expression showed robust diagnostic capability for osteosarcoma, suggesting potential as a biomarker

  • Molecular mechanisms in tumor progression:

    • In vitro studies revealed that elevating ACTR2 or suppressing miR-374a-5p attenuated proliferation, invasion, migration, and epithelial-mesenchymal transition (EMT) of osteosarcoma cells while enhancing their apoptosis

    • Conversely, upregulation of miR-374a-5p yielded opposing effects, suggesting a regulatory axis

    • These findings position ACTR2 as a potential tumor suppressor in certain cancer contexts

• ACTR2 in cell migration disorders:

  • Fundamental mechanisms:

    • The Arp2/3 complex mediates actin polymerization, providing force for cell motility

    • Silencing almost any subunit of the Arp2/3 complex leads to significant decrease in cell migratory capability

  • Applications in disease research:

    • ACTR2 antibodies enable visualization of aberrant cytoskeletal architecture in migration-related disorders

    • Immunofluorescence studies using ACTR2 antibodies have helped characterize cell motility defects in various pathological conditions

• Role in DNA repair and genomic stability:

  • Nuclear functions of ACTR2:

    • Beyond cytoplasmic roles, ACTR2 promotes nuclear actin polymerization