ACTR3B Antibody

What is ACTR3B and what cellular functions does it regulate?

ACTR3B is a 418 amino acid protein with an observed molecular weight of approximately 48 kDa that functions as an actin-related protein homologous to yeast ARP3 . It is a vital component of actin cytoskeletal regulation, playing crucial roles in cell morphology, migration, and invasion. ACTR3B shares structural and functional similarities with ACTR3, which has been demonstrated to be a component of the Arp2/3 complex that regulates actin polymerization and filament formation . In cellular contexts, ACTR3B contributes to the formation of lamellipodia and filopodia, which are critical for cell movement. Research has shown that related proteins in this family can combine with other factors like profilin-1 to regulate these cellular structures with assistance from proteins such as LIM domain only 2 (LMO2) .

What are the primary applications for ACTR3B antibodies in research?

ACTR3B antibodies are primarily utilized in several key applications:

• Western Blotting (WB): Used for protein detection and quantification, with recommended dilutions ranging from 1:5000 to 1:50000 depending on the specific antibody and sample type .

• Immunohistochemistry (IHC): Applied for tissue localization studies with typical dilutions of 1:500 to 1:2000 .

• ELISA: Employed for quantitative protein analysis in various sample types .

These applications enable researchers to investigate ACTR3B expression levels, protein localization, and interactions with other cellular components. When designing experiments, it's essential to validate the antibody in your specific biological system, as reactivity can vary across species and tissues.

How do I select the appropriate ACTR3B antibody for my research?

Selection of an appropriate ACTR3B antibody should be based on several methodological considerations:

• Target epitope region: Different antibodies target specific amino acid regions of ACTR3B (e.g., AA 1-418, AA 189-418, AA 325-352) . Consider which domain is most relevant to your research question.

• Species reactivity: Verify the antibody's documented reactivity with your experimental species. Available antibodies show varying reactivity profiles:

  • Human-only reactivity

  • Human and mouse reactivity

  • Broad reactivity across multiple species including human, mouse, rat, cow, dog, guinea pig, zebrafish, horse, rabbit, monkey, etc.

• Antibody type and host:

  • Polyclonal vs. monoclonal: Polyclonal antibodies offer broader epitope recognition but potentially lower specificity; monoclonal antibodies provide higher specificity to a single epitope .

  • Host species (mouse, rabbit): Consider compatibility with your secondary detection systems and other antibodies in multiplex experiments .

• Validated applications: Ensure the antibody has been validated for your specific application (WB, IHC, ELISA) .

What are the optimal protocols for using ACTR3B antibodies in Western blotting?

Western blotting with ACTR3B antibodies requires careful optimization of several parameters:

Sample preparation:

• Brain tissue samples from various species (human, mouse, rat, pig, rabbit) and cell lines (MOLT-4, Jurkat, K-562) have been successfully used with ACTR3B antibodies .

• Proper lysis buffers should contain protease inhibitors to prevent degradation.

Gel electrophoresis and transfer:

• Since ACTR3B has a molecular weight of approximately 48 kDa, use appropriate percentage gels (10-12%) for optimal resolution .

• Transfer conditions may need adjustment based on protein size and hydrophobicity.

Antibody incubation:

• Primary antibody: Use dilutions between 1:5000 and 1:50000 depending on the specific antibody and detection system .

• Secondary antibody: Select based on the host species of your primary antibody (anti-mouse for mouse-derived antibodies, anti-rabbit for rabbit-derived antibodies).

The specificity of detection should be verified by appropriate controls, including a loading control and, ideally, samples with known expression levels or knockdown/knockout samples. Observed ACTR3B bands should appear at approximately 48 kDa .

How should I optimize immunohistochemistry protocols for ACTR3B detection in tissue samples?

For optimal immunohistochemical detection of ACTR3B:

Tissue preparation and antigen retrieval:

• Fixed tissues (preferably formalin-fixed, paraffin-embedded) should undergo appropriate antigen retrieval.

• TE buffer at pH 9.0 is the suggested retrieval method, though citrate buffer at pH 6.0 may serve as an alternative .

Antibody application:

• Use dilutions between 1:500 and 1:2000 for primary ACTR3B antibodies .

• Brain tissue samples have shown positive IHC results and can serve as positive controls .

Signal development and interpretation:

• Choose visualization systems compatible with your primary antibody host species.

• Include appropriate controls (negative control without primary antibody and positive control tissues).

It's important to note that antibody performance may vary between tissue types and fixation methods, requiring optimization for each specific application. Mouse brain tissue has shown reliable positive staining and can serve as an effective control tissue .

What are the major challenges in reproducing ACTR3B antibody experiments and how can I address them?

Common challenges and their methodological solutions include:

• Inconsistent antibody performance:

  • Solution: Perform thorough validation using multiple detection methods. Compare results from different antibody clones or sources.

  • Implement careful antibody aliquoting and storage at -20°C to maintain stability. Note that ACTR3B antibodies are typically stable for one year after shipment when properly stored .

• Non-specific binding:

  • Solution: Optimize blocking conditions using BSA or non-fat milk.

  • Increase washing steps and duration.

  • Consider using more specific monoclonal antibodies if polyclonal antibodies show high background.

• Variability in ACTR3B expression levels:

  • Solution: Establish standardized positive controls for your experimental system.

  • Understand that ACTR3B expression can vary significantly between different cell types and tissues, with brain tissue consistently showing detectable expression .

• Cross-reactivity with related proteins:

  • Solution: Conduct specificity tests using competing peptides or lysates from knockdown/knockout systems.

  • Select antibodies that have been validated for specificity to human ACTR3B/ARP4 rather than detecting the related ACTR3 protein .

How can ACTR3B antibodies be employed to investigate cytoskeletal dynamics and cell migration?

ACTR3B, like its homolog ACTR3, plays crucial roles in actin cytoskeletal organization that affects cell morphology and motility. To investigate these processes:

• Immunofluorescence colocalization studies:

  • Use ACTR3B antibodies in combination with F-actin staining (phalloidin) to visualize colocalization at the leading edge of migrating cells.

  • Related research has shown that knockdown of the homologous ACTR3 alters F-actin distribution, suggesting ACTR3B may play similar roles .

• Live-cell imaging:

  • Combine antibody-based detection methods with GFP-tagged ACTR3B constructs to track dynamic changes during cell migration.

  • Focus on lamellipodia and filopodia formation, as these structures are regulated by Arp2/3 complex proteins.

• Wound healing and invasion assays:

  • Apply ACTR3B antibodies to monitor protein expression and localization during cell migration in wound healing assays.

  • Research on ACTR3 has demonstrated that knockdown significantly inhibits cell migration and invasion in transwell assays, suggesting similar approaches would be valuable for ACTR3B studies .

• Pull-down assays:

  • Use ACTR3B antibodies to identify interaction partners that regulate cytoskeletal dynamics.

  • Consider examining interactions with known actin-regulatory proteins such as profilin-1 and components of the Arp2/3 complex.

Research findings with the related ACTR3 protein showed that knockdown significantly repressed migration and invasion of pancreatic cancer cells, suggesting ACTR3B may have similar functions worth investigating .

What methodological approaches can be used to study ACTR3B in the context of epithelial-mesenchymal transition (EMT)?

Based on findings with the homologous ACTR3 protein in pancreatic cancer, several methodological approaches can be adapted for ACTR3B studies in EMT:

• Expression correlation analysis:

  • Use ACTR3B antibodies to quantify protein levels in relation to established EMT markers (E-cadherin, N-cadherin, vimentin) via western blotting and immunohistochemistry.

  • Research on ACTR3 demonstrated altered expression of EMT markers when the protein was knocked down .

• Knockdown/overexpression studies:

  • Generate ACTR3B knockdown or overexpression models and assess changes in:
    a) EMT marker expression
    b) Cell morphology (using ACTR3B antibodies to confirm altered expression)
    c) Migration and invasion capabilities

• Signaling pathway analysis:

  • Combine ACTR3B antibody detection with phospho-specific antibodies for EMT-related signaling molecules.

  • Consider examining potential regulatory relationships with transcription factors that control EMT programs.

• In vivo metastasis models:

  • Use ACTR3B antibodies to assess protein expression in primary tumors versus metastatic sites.

  • Based on ACTR3 research, which identified it as a metastasis-associated gene in pancreatic cancer, similar approaches may be valuable for ACTR3B .

ACTR3 research revealed significant associations between expression levels and invasive properties of cancer cells, with knockdown experiments demonstrating reduced migration and invasion capabilities . These findings suggest ACTR3B may play comparable roles worth investigating through similar methodological approaches.

How can I investigate potential differential functions between ACTR3 and ACTR3B in cellular processes?

To distinguish between the related but potentially functionally distinct ACTR3 and ACTR3B proteins:

• Selective knockdown experiments:

  • Design specific siRNAs or CRISPR-Cas9 guides targeting either ACTR3 or ACTR3B.

  • Use antibodies specific to each protein to confirm selective knockdown.

  • Compare phenotypes resulting from individual and combined knockdowns.

• Co-immunoprecipitation studies:

  • Use specific antibodies against ACTR3 and ACTR3B to identify unique interaction partners.

  • Compare protein complexes associated with each protein to identify distinct functional roles.

• Tissue and cell type expression profiling:

  • Apply antibodies against both proteins across tissue panels to identify differential expression patterns.

  • ACTR3B antibodies have shown reactivity in brain tissue samples from multiple species, which may indicate tissue-specific roles .

• Rescue experiments:

  • In knockdown models of either protein, attempt rescue with the other protein to identify redundant versus unique functions.

  • Use antibodies to confirm expression of the rescue construct.

This comparative approach is particularly important since the literature suggests potentially overlapping yet distinct functions, with ACTR3 being well-studied in contexts like pancreatic cancer progression, while ACTR3B's specific roles remain less characterized .

What is the significance of ACTR3B expression in cancer research and how can antibodies help investigate this?

While direct evidence for ACTR3B in cancer is more limited in the provided search results, insights can be drawn from studies of the related ACTR3 protein and methodological approaches can be adapted for ACTR3B:

• Expression analysis in tumor tissues:

  • Use ACTR3B antibodies for immunohistochemical staining of tumor microarrays to assess expression patterns across cancer types and stages.

  • Studies of ACTR3 showed significantly increased expression in pancreatic ductal adenocarcinoma (PDAC) tissues compared to non-cancerous tissues, with a 7-fold higher expression in tumor samples .

• Prognostic correlations:

  • Apply ACTR3B antibodies in tissue studies correlated with patient outcome data.

  • Research on ACTR3 demonstrated that higher expression was predictive of poor outcomes for PDAC patients, suggesting similar investigations may be valuable for ACTR3B .

• Functional studies in cancer cell lines:

  • Use ACTR3B antibodies to monitor protein levels in knockdown or overexpression experiments.

  • Assess effects on cancer hallmarks such as proliferation, migration, invasion, and resistance to therapy.

  • Studies with ACTR3 showed that knockdown significantly inhibited invasive and migratory capacity of cancer cells .

• Potential therapeutic target assessment:

  • Investigate ACTR3B as a potential therapeutic target in cancer models, similar to how ACTR3 has been identified as a potential therapeutic target for PDAC metastasis .

The homologous ACTR3 protein has been found to be upregulated in several cancer types, including gastric cancer, squamous cell carcinoma, colorectal cancer, and pancreatic cancer . These findings suggest ACTR3B may also have significant roles in cancer processes worthy of investigation.

How can I troubleshoot ACTR3B antibody performance in different sample types?

When encountering difficulties with ACTR3B antibody performance across different sample types:

• Sample-specific optimization:

  • For cell lines: MOLT-4, Jurkat, and K-562 cells have shown positive Western blot results with ACTR3B antibodies and can serve as positive controls .

  • For tissue samples: Brain tissue from multiple species (mouse, rat, pig, rabbit) consistently shows detectable ACTR3B expression and can function as reliable positive controls .

• Antigen retrieval modification:

  • For IHC applications, compare TE buffer (pH 9.0) versus citrate buffer (pH 6.0) for optimal epitope exposure .

  • Different fixation methods may require different retrieval approaches.

• Antibody dilution optimization:

  • Perform titration experiments across a range of dilutions:

    • For Western blot: Test dilutions from 1:5000 to 1:50000

    • For IHC: Test dilutions from 1:500 to 1:2000

• Species-specific considerations:

  • Select antibodies with validated reactivity for your species of interest.

  • Some ACTR3B antibodies show broad cross-reactivity across species (human, mouse, rat, pig, rabbit), while others are more restricted .

If detecting ACTR3B in a previously untested sample type, begin with conditions optimized for brain tissue, as this has been consistently successful across multiple species .

What are the latest methodological advances in investigating ACTR3B's role in cytoskeletal regulation?

Advanced approaches for investigating ACTR3B in cytoskeletal regulation include:

• Super-resolution microscopy techniques:

  • Apply techniques such as STORM, PALM, or SIM to visualize ACTR3B localization within actin structures at nanometer resolution.

  • Combine ACTR3B antibody staining with actin and other cytoskeletal markers.

• Live-cell dynamics analysis:

  • Use fluorescently tagged ACTR3B constructs to monitor dynamic recruitment to actin structures.

  • Validate observations using fixed-cell immunofluorescence with ACTR3B antibodies.

• Proximity labeling methods:

  • Apply BioID or APEX2 proximity labeling to identify proteins in close spatial proximity to ACTR3B.

  • Validate interactions using co-immunoprecipitation with ACTR3B antibodies.

• Quantitative phosphoproteomics:

  • Investigate potential regulatory phosphorylation of ACTR3B and its impact on cytoskeletal functions.

  • Use phospho-specific antibodies if available, or analyze immunoprecipitated ACTR3B by mass spectrometry.

Research on related proteins has demonstrated that Arp2/3 complex components function as actin-regulatory proteins that affect cell morphology and mobility, suggesting similar roles for ACTR3B worthy of investigation through these advanced approaches .

What critical controls should I include when using ACTR3B antibodies in my experiments?

Implementing appropriate controls is essential for reliable ACTR3B antibody-based experiments:

• Positive controls:

  • Tissue samples: Brain tissue from multiple species (mouse, rat, pig, rabbit) has shown reliable ACTR3B expression .

  • Cell lines: MOLT-4, Jurkat, and K-562 cells have demonstrated detectable ACTR3B expression in Western blot applications .

• Negative controls:

  • Primary antibody omission control: Process samples without primary antibody to assess secondary antibody specificity.

  • Knockdown/knockout validation: Where possible, include ACTR3B-depleted samples to confirm antibody specificity.

• Loading and processing controls:

  • For Western blots: Include housekeeping protein controls (β-actin, GAPDH, etc.)

  • For IHC/IF: Include internal control tissues on the same slide.

• Antibody specificity controls:

  • Peptide competition assay: Pre-incubate antibody with immunizing peptide to block specific binding.

  • Multiple antibody validation: When possible, confirm results using antibodies targeting different epitopes of ACTR3B (e.g., N-terminal vs. C-terminal) .

These controls help ensure that observed signals are specific to ACTR3B rather than resulting from non-specific binding or technical artifacts.

How do I interpret conflicting results from different ACTR3B antibodies?

When faced with discrepant results using different ACTR3B antibodies:

• Epitope mapping analysis:

  • Compare the target regions of each antibody (AA 1-418, AA 189-418, AA 325-352, etc.) .

  • Different epitopes may be differentially accessible depending on protein conformation, interactions, or post-translational modifications.

• Methodological validation:

  • Verify antibody specificity using multiple approaches:

    • Western blot for size verification (expected 48 kDa)

    • Immunoprecipitation followed by mass spectrometry

    • siRNA knockdown to confirm signal reduction

• Isoform consideration:

  • Investigate whether antibodies might detect different isoforms or splice variants of ACTR3B.

  • Compare antibody binding sites to known isoform sequences.

• Cross-reactivity assessment:

  • Evaluate potential cross-reactivity with related proteins, particularly ACTR3.

  • Some antibodies are specifically validated against human ACTR3B/ARP4, which may help avoid cross-reactivity issues .

What are the best practices for storing and handling ACTR3B antibodies to maintain optimal performance?

To ensure consistent ACTR3B antibody performance over time:

Following these storage and handling practices helps ensure reproducible experimental results when working with ACTR3B antibodies.