ARPC1B Antibody

What is ARPC1B and what is its biological significance?

ARPC1B (also known as ARC41, p41-ARC, or p40-ARC) is one of seven subunits of the human Arp2/3 protein complex and belongs to the SOP2 family. The protein plays a crucial role in actin cytoskeletal dynamics through the regulation of actin filament branching. ARPC1B is primarily expressed in blood cells, suggesting its specialized function in the hematopoietic system .

From a biological perspective, ARPC1B is essential for multiple cellular processes, including:

• Formation of immune synapses in T cells

• Endocytosis and phagocytosis in antigen-presenting cells

• Maintaining centrosomal homeostasis

• Regulation of mitotic integrity in mammalian cells

Notably, ARPC1B has been identified as both a physiological activator and substrate of Aurora A kinase, and these interactions are critical for maintaining mitotic integrity in mammalian cells .

How do ARPC1B antibodies differ in their detection applications?

ARPC1B antibodies are available in different formats optimized for specific experimental applications. Based on the search results, commercially available ARPC1B antibodies exhibit the following application profiles:

The observed molecular weight of ARPC1B is approximately 37-38 kDa in experimental settings, which differs slightly from the calculated molecular weight of 41 kDa. This discrepancy may be due to post-translational modifications or protein processing and should be considered when interpreting Western blot results .

What is the recommended methodology for antigen retrieval when using ARPC1B antibodies in immunohistochemistry?

For optimal immunohistochemical detection of ARPC1B in tissue samples, proper antigen retrieval is essential. Based on the available data, the following protocol is recommended:

Primary method: Antigen retrieval with TE buffer (pH 9.0). This alkaline buffer system has been validated for detecting ARPC1B in multiple tissue types including human colon cancer tissue, human lung cancer tissue, mouse spleen tissue, and human appendicitis tissue .

Alternative method: If the primary method yields suboptimal results, citrate buffer (pH 6.0) can be used as an alternative antigen retrieval solution.

The choice between these two methods may depend on:

• The specific tissue being examined

• Fixation conditions

• The particular antibody clone being used

• The presence of potential cross-reactive antigens

Researchers should optimize antigen retrieval conditions for their specific experimental system to achieve the highest signal-to-noise ratio .

How can ARPC1B antibodies be validated for specificity in experimental systems?

Validating antibody specificity is crucial for generating reliable scientific data. For ARPC1B antibodies, the following validation approaches are recommended:

• Positive control testing: Confirmed detection in HeLa cells, HEK-293T cells, Jurkat cells, K-562 cells, THP-1 cells, and rat spleen tissues has been reported. These can serve as appropriate positive controls for Western blotting experiments .

• Negative control testing: Include samples with ARPC1B knockdown or knockout. The specificity of ARPC1B knockdown can be verified by probing for other Arp2/3 complex components such as Arpc2 and Arp3, which should remain unchanged if the knockdown is specific to ARPC1B .

• Co-immunoprecipitation analysis: To validate the antibody's ability to detect native protein complexes, immunoprecipitation followed by Western blotting can be performed. For instance, γ-tubulin has been shown to co-immunoprecipitate with endogenous ARPC1B and Aurora A, providing a useful positive control for such experiments .

• Cross-reactivity assessment: When studying related proteins, it's important to ensure the antibody doesn't cross-react with similar proteins. For ARPC1B, particular attention should be paid to potential cross-reactivity with ARPC1A, the other isoform of ARPC1 .

What is the relationship between ARPC1B deficiency and immune dysfunction?

ARPC1B deficiency represents a recently characterized syndrome of combined immune deficiency associated with significant clinical implications. Research has revealed that homozygous mutations in the ARPC1B gene cause an autosomal recessive syndrome characterized by:

• Combined immune deficiency with recurrent infections

• Impaired T-cell migration and proliferation

• Elevated levels of immunoglobulin E (IgE) and immunoglobulin A (IgA)

• Allergic manifestations and asthma

• Autoimmunity and autoinflammatory processes

• In some cases, thrombocytopenia

The mechanistic basis for these manifestations stems from ARPC1B's crucial role in actin cytoskeletal dynamics. Actin and actin-regulating proteins control diverse immune processes, including cellular infrastructure, motility, signaling, and vesicle transport. Specifically, ARPC1B is critical for:

• Formation of the immune synapse in T cells

• Endocytosis and phagocytosis in antigen-presenting cells

• B-cell receptor signaling, with defects potentially leading to altered B-cell tolerance and autoimmunity

Interestingly, a novel synonymous variant (c.783G>A, p.Ala261Ala) in the ARPC1B gene has been found to affect mRNA splicing, resulting in reduced levels of normal ARPC1B transcripts. This finding highlights the importance of investigating synonymous mutations that may have pathogenic consequences through altered splicing mechanisms .

How does ARPC1B localization relate to its function in cellular processes?

ARPC1B exhibits specific subcellular localization patterns that are directly related to its diverse functions:

• Centrosomal localization: ARPC1B has been identified as a centrosomal protein with a distinct role in centrosomal homeostasis. Unlike other components of the Arp2/3 complex (such as Arp3), ARPC1B specifically localizes to centrosomes, suggesting it may have functions independent of the canonical Arp2/3 complex .

• Association with Aurora A kinase: Co-immunoprecipitation studies have demonstrated that ARPC1B interacts with Aurora A kinase, a key regulator of mitotic progression. This interaction appears to be specific, as Aurora A does not co-immunoprecipitate with Arp3. This suggests that a fraction of ARPC1B exists independently of the Arp2/3 complex and is involved in mitotic regulation through its interaction with Aurora A .

• Interaction with γ-tubulin: ARPC1B has been shown to co-immunoprecipitate with γ-tubulin in metaphase-arrested cells, further supporting its role in centrosome function and mitotic spindle organization .

The dual localization of ARPC1B in both cytoskeletal structures and centrosomes makes it a unique component of the actin regulation machinery, with implications for both immune cell function and cell division. Researchers investigating ARPC1B should consider these distinct localizations when designing imaging experiments and interpreting subcellular distribution patterns .

How does ARPC1B deficiency impact DNA damage response and radiosensitivity?

Recent research has uncovered a previously unrecognized connection between ARPC1B deficiency and increased radiosensitivity (RS). This finding adds a new dimension to our understanding of the clinical phenotype associated with ARPC1B mutations. The evidence for increased RS in ARPC1B-deficient patients includes:

• Increased chromosomal aberrations: Higher levels of chromatid-type aberrations were observed in cells from ARPC1B-deficient patients after exposure to ionizing radiation or treatment with the radiomimetic agent bleomycin .

• Persistent DNA damage signaling: Enhanced γH2AX foci formation, a marker of DNA double-strand breaks, was detected in ARPC1B-deficient cells following radiation exposure .

• Cell cycle abnormalities: An increased number of cells arrested in the G2/M phase of the cell cycle was observed in ARPC1B-deficient cells after radiation exposure, suggesting defects in DNA damage checkpoint resolution .

These findings suggest that ARPC1B may play a previously unappreciated role in the DNA damage response pathway. The mechanism linking cytoskeletal dynamics to DNA repair remains to be fully elucidated, but may involve:

• Nuclear actin dynamics affecting chromatin remodeling during DNA repair

• Altered cellular transport of DNA repair factors

• Dysregulation of cell cycle checkpoints due to centrosomal abnormalities

From a clinical perspective, the increased radiosensitivity in ARPC1B-deficient patients has important implications for their management, particularly regarding diagnostic radiation exposure and therapeutic interventions that may cause DNA damage .

What are the technical challenges in distinguishing between ARPC1B-dependent and Arp2/3 complex-dependent functions?

A significant challenge in ARPC1B research is distinguishing between functions that depend specifically on ARPC1B versus those that require the intact Arp2/3 complex. Evidence suggests that ARPC1B may have both Arp2/3-dependent and Arp2/3-independent functions:

• Experimental evidence for independent functions: Co-immunoprecipitation studies have shown that γ-tubulin and Aurora A interact with ARPC1B but not with Arp3, suggesting that a fraction of ARPC1B exists independently of the canonical Arp2/3 complex .

• Methodological considerations:

  • When investigating ARPC1B by immunoprecipitation, researchers should consider whether the Arp2/3 complex might dissociate during experimental procedures

  • Control experiments should include blotting for other Arp2/3 complex components to determine if the complex remains intact

  • Comparison of phenotypes between ARPC1B knockdown and knockdown of other Arp2/3 complex components can help distinguish ARPC1B-specific functions

• Alternative experimental approaches:

  • Domain-specific mutagenesis to disrupt ARPC1B interactions with specific partners while preserving others

  • Proximity labeling techniques (BioID, APEX) to identify context-specific interaction partners

  • Live-cell imaging with differentially labeled Arp2/3 components to track complex assembly/disassembly

When designing experiments to study ARPC1B functions, researchers should carefully consider whether their hypothesis involves ARPC1B as part of the Arp2/3 complex or in its potential independent roles, particularly in centrosome function and mitotic regulation .

What are the methodological considerations for studying novel ARPC1B mutations and their impact on protein function?

The identification of novel ARPC1B mutations, including synonymous mutations that affect splicing, presents unique challenges for researchers. Here are key methodological considerations:

• Comprehensive mutation analysis approaches:

  • Next-generation sequencing (NGS) approaches, particularly trio-based sequencing (proband and parents), have proven valuable for identifying novel ARPC1B variants

  • RNA studies are essential for evaluating the impact of synonymous mutations on splicing, as demonstrated by the c.783G>A (p.Ala261Ala) variant that affects mRNA processing

  • Protein expression analysis using Western blotting with validated antibodies is crucial to confirm the effect of mutations on ARPC1B protein levels

• Functional validation strategies:

  • Analysis of T-cell migration and proliferation assays to assess immune cell function

  • Evaluation of actin polymerization using fluorescence-based assays

  • Assessment of B-cell receptor signaling and tolerance mechanisms

  • Measurement of immunoglobulin levels, particularly IgE and IgA

  • DNA damage response assays, including γH2AX foci formation after radiation exposure

• Clinical correlations:

  • Detailed phenotyping of patients with ARPC1B mutations reveals significant intrafamilial clinical heterogeneity

  • The absence of certain features (e.g., thrombocytopenia) in some patients with ARPC1B mutations suggests genotype-phenotype correlations that require further investigation

  • The discovery of increased radiosensitivity as a novel feature of ARPC1B deficiency highlights the importance of comprehensive phenotyping

This methodological framework can guide researchers investigating novel ARPC1B mutations and help establish causality between genetic variants and clinical phenotypes.

What are the optimal conditions for Western blot analysis using ARPC1B antibodies?

Successful Western blot detection of ARPC1B requires careful optimization of experimental conditions. Based on validated protocols, the following methodological considerations are recommended:

• Sample preparation:

  • Cell lysis should be performed using buffers that preserve protein-protein interactions if studying ARPC1B complexes

  • Positive controls should include HeLa cells, HEK-293T cells, Jurkat cells, K-562 cells, or THP-1 cells, which have confirmed ARPC1B expression

  • Protein loading amounts typically range from 10-30 μg per lane, depending on expression levels

• Antibody selection and dilution:

  • For rabbit polyclonal antibody (28368-1-AP): use at 1:1000-1:6000 dilution

  • For mouse monoclonal antibody (67320-1-Ig): use at 1:5000-1:50000 dilution

  • The wide dilution ranges indicate that optimization is necessary for each experimental system

• Detection considerations:

  • The observed molecular weight of ARPC1B is approximately 37-38 kDa, which differs from the calculated 41 kDa

  • Secondary antibody selection should match the host species (anti-rabbit or anti-mouse)

  • When studying ARPC1B in the context of the Arp2/3 complex, consider multiplexing with antibodies against other complex components (e.g., Arpc2, Arp3) to assess complex integrity

• Troubleshooting guidance:

  • If multiple bands are observed, validation with ARPC1B knockdown controls is recommended

  • For weak signals, extended exposure times or signal enhancement systems may be necessary

  • When comparing ARPC1B levels across samples, normalization to loading controls is essential

These methodological details are critical for generating reproducible and interpretable Western blot data when studying ARPC1B.

How can ARPC1B antibodies be effectively used in immunohistochemistry studies?

Immunohistochemical detection of ARPC1B requires specific methodological considerations to achieve optimal staining with minimal background. Based on validated protocols, the following approach is recommended:

• Tissue preparation and fixation:

  • Formalin-fixed, paraffin-embedded (FFPE) tissues have been successfully used for ARPC1B detection

  • Positive control tissues should include spleen (mouse or human), colon cancer tissue, lung cancer tissue, or appendicitis tissue

  • Section thickness typically ranges from 4-6 μm

• Antigen retrieval methods:

  • Primary method: TE buffer (pH 9.0)

  • Alternative method: Citrate buffer (pH 6.0)

  • Heating methods may include microwave, pressure cooker, or water bath, with optimization necessary for each system

• Antibody application:

  • For rabbit polyclonal antibody (28368-1-AP): use at 1:250-1:1000 dilution

  • For mouse monoclonal antibody (67320-1-Ig): use at 1:500-1:2000 dilution

  • Incubation conditions typically involve overnight incubation at 4°C or 1-2 hours at room temperature

  • Detection systems should be compatible with the primary antibody species

• Interpretation guidance:

  • ARPC1B typically shows cytoplasmic staining pattern

  • In certain cell types, centrosomal localization may be observed

  • When studying ARPC1B-deficient patient samples, residual staining should be carefully evaluated to determine if it represents true ARPC1B expression or background

These methodological details can help researchers design effective immunohistochemistry experiments to study ARPC1B expression in normal and pathological tissues.

What are the best practices for immunoprecipitation of ARPC1B and its interaction partners?

Immunoprecipitation (IP) is a valuable technique for studying ARPC1B and its protein interactions. Based on the available data, the following methodology is recommended:

• Lysate preparation:

  • Use lysis buffers that preserve protein-protein interactions (e.g., NP-40 or CHAPS-based buffers)

  • Typical protein concentrations range from 1.0-3.0 mg total protein lysate

  • Pre-clearing of lysates with control IgG and protein A/G beads can reduce non-specific binding

• Antibody amounts and conditions:

  • For rabbit polyclonal antibody (28368-1-AP): use 0.5-4.0 μg antibody per 1.0-3.0 mg of total protein lysate

  • Incubation is typically performed overnight at 4°C with gentle rotation

  • Protein A/G beads are commonly used for capturing antibody-antigen complexes

• Co-immunoprecipitation considerations:

  • When studying ARPC1B interactions, validated partners include γ-tubulin and Aurora A

  • Controls should include immunoprecipitation with non-specific IgG

  • When investigating Arp2/3 complex integrity, blotting for other complex components (e.g., Arp3, Arpc2) is recommended

• Validation approaches:

  • Reciprocal co-immunoprecipitation experiments (e.g., IP with anti-Aurora A followed by ARPC1B detection) can strengthen interaction findings

  • Comparison of interactions under different cellular conditions (e.g., different cell cycle phases) can provide insight into context-dependent interactions

  • For novel interaction partners, confirmation with alternative techniques like proximity ligation assay is recommended

These methodological details can guide researchers in designing effective immunoprecipitation experiments to study ARPC1B interactions and complex formation.