ARPC1B Antibody

What is ARPC1B and why is it important in research?

ARPC1B (Actin Related Protein 2/3 Complex, Subunit 1B, 41kDa) is a critical component of the Arp2/3 complex, which functions as a nucleation factor for generating branched F-actin networks in cells. ARPC1B plays essential roles in cytoskeletal reorganization, particularly in immune cells such as cytotoxic T lymphocytes (CTLs). Its importance in research stems from its involvement in immune synapse formation, cell migration, and maintenance of key membrane proteins. Deficiency in ARPC1B has been linked to combined immunodeficiency with symptoms of immune dysregulation, including recurrent viral infections and reduced CD8+ T cell counts . This makes ARPC1B antibodies valuable tools for investigating cytoskeletal dynamics and immune cell function in both normal and pathological conditions. Understanding ARPC1B function provides insights into fundamental cellular processes and potential therapeutic targets for immunological disorders.

What types of ARPC1B antibodies are available for research applications?

ARPC1B antibodies come in various formats to suit different research applications. The primary types include:

• Host species variations: Predominantly rabbit polyclonal antibodies, though goat polyclonal options are also available .

• Target region specificity:

  • N-terminal targeting antibodies

  • Antibodies targeting specific amino acid regions (e.g., AA 16-316, AA 159-188, AA 322-372)

• Conjugation options:

  • Unconjugated primary antibodies

  • HRP-conjugated for enhanced chemiluminescent detection

  • FITC-conjugated for fluorescence applications

  • Biotin-conjugated for streptavidin-based detection systems

Each antibody type has specific reactivity profiles, with most showing reactivity to human ARPC1B, while some also cross-react with mouse and rat homologs. The choice of antibody depends on the planned experimental technique, target species, and required sensitivity.

What are the standard applications for ARPC1B antibodies?

ARPC1B antibodies can be employed in multiple experimental techniques, each providing different insights into protein expression, localization, and function:

Research has utilized these techniques to demonstrate ARPC1B's roles in centrosomal functions, Aurora A activation, and immune synapse formation . For example, immunoprecipitation followed by Western blotting revealed ARPC1B's interaction with Aurora A in a cell cycle-dependent manner . Similarly, superresolution microscopy of immunofluorescence-labeled cytotoxic T lymphocytes has demonstrated ARPC1B's role in lamellipodia formation and actin reorganization at the immune synapse .

How does ARPC1B function independently of the Arp2/3 complex?

While ARPC1B is traditionally recognized as a component of the Arp2/3 complex, research has revealed that it also functions independently. Sucrose gradient sedimentation experiments using ZR-75 cell extracts have demonstrated that ARPC1B exists in two distinct peaks: fractions 6-9 (representing stand-alone ARPC1B) and fractions 12-15 (representing ARPC1B as part of the Arp2/3 complex) . This dual existence has significant functional implications.

The stand-alone ARPC1B interacts with Aurora A kinase and γ-tubulin, independent of other Arp2/3 complex components like Arp3. This was confirmed when immunoprecipitation using an anti-Aurora A antibody failed to pull down Arp3, while successfully co-immunoprecipitating ARPC1B . Furthermore, ARPC1B enhances Aurora A kinase activity in vitro, while neither Arp3 nor the related ARPC1A subunit showed this ability. These findings indicate that ARPC1B has functions beyond its structural role in the Arp2/3 complex, particularly in centrosomal dynamics and mitotic processes.

When designing experiments to study ARPC1B, researchers should consider this dual functionality and employ approaches that can distinguish between ARPC1B as part of the Arp2/3 complex versus its independent roles. This might include using specific antibodies that recognize different conformational states or employing size-exclusion chromatography to separate the different complexes.

What are the optimal methods for investigating ARPC1B's role in immune synapse formation?

Investigating ARPC1B's role in immune synapse formation requires a multi-faceted approach that captures both structural and functional aspects of this dynamic process. Based on published research methodologies, the following approaches are recommended:

• High-resolution imaging techniques:

  • Superresolution microscopy of phalloidin-labeled CTLs allows visualization of F-actin structures at the immune synapse

  • Electron microscopy provides detailed visualization of membrane interdigitations at the CTL-target cell interface

  • Live cell imaging with fluorescent probes (e.g., mApple-Lifeact) enables real-time tracking of actin dynamics during synapse formation

• Immunofluorescence co-localization studies:

  • Use anti-ARPC1B antibodies in combination with phalloidin staining to simultaneously visualize ARPC1B and F-actin distribution at the immune synapse

  • Include markers for other immune synapse components (e.g., perforin, LFA-1) to understand spatial relationships

• Functional assays:

  • Cytotoxicity assays to correlate ARPC1B localization with CTL killing efficiency

  • TCR signaling measurements to link ARPC1B-dependent actin reorganization with T cell activation

Studies comparing healthy donor CTLs with ARPC1B-deficient CTLs have revealed that ARPC1B is essential for the formation of actin-rich interdigitations at the immune synapse . In ARPC1B-deficient CTLs, synapses appear flattened with reduced actin accumulation, correlating with defective cytolytic function. This highlights the importance of using both structural and functional readouts when investigating ARPC1B's role at the immune synapse.

How can ARPC1B antibodies be used to study the mechanisms of ARPC1B deficiency-related immunodeficiency?

ARPC1B deficiency leads to combined immunodeficiency with symptoms including recurrent viral infections and reduced CD8+ T cell counts. Studying the mechanisms underlying this condition requires sophisticated experimental approaches using ARPC1B antibodies:

• Patient-derived cell analysis:

  • Western blotting with ARPC1B antibodies can confirm protein deficiency in patient samples

  • Parallel probing for ARPC1A can reveal compensatory upregulation, as observed in multiple studies

• Receptor trafficking studies:

  • Immunofluorescence and biochemical fractionation can track the localization and recycling of key receptors (TCR, CD8, GLUT1) that are impaired in ARPC1B deficiency

  • Co-staining for retromer and WASH complex components can reveal defects in the recycling machinery

• Cytoskeletal dynamics assessment:

  • Super-resolution microscopy with phalloidin and anti-ARPC1B staining can visualize lamellipodia formation defects

  • Live-cell imaging using actin probes (e.g., Lifeact) in combination with immunolabeling can correlate ARPC1B localization with dynamic actin structures

• Functional immunological assays:

  • T cell proliferation assays correlated with ARPC1B levels can link protein expression to functional outcomes

  • Target cell killing assays with immunofluorescence to simultaneously assess synapse formation and cytolytic function

Research has revealed that ARPC1B is indispensable for CTL cytotoxicity at multiple levels. First, it's essential for lamellipodia formation, cell migration, and actin reorganization across the immune synapse. Second, ARPC1B is critical for maintaining TCR, CD8, and GLUT1 membrane proteins at the plasma membrane, as recycling via the retromer and WASH complexes is impaired in its absence . These defects lead to compromised T cell signaling and proliferation, explaining the progressive loss of CD8+ T cells observed in patients.

What are the key considerations for validating ARPC1B antibody specificity?

Thorough validation of ARPC1B antibody specificity is crucial for ensuring reliable experimental results, particularly given the existence of the similar ARPC1A isoform. A comprehensive validation strategy should include:

• Genetic controls:

  • Testing the antibody in ARPC1B-deficient patient samples or ARPC1B knockout cell lines

  • Using siRNA knockdown to create samples with reduced ARPC1B expression

  • Testing in cells overexpressing tagged ARPC1B to confirm detection

• Biochemical validation:

  • Western blot analysis to confirm a single band at the expected molecular weight (41 kDa)

  • Peptide competition assays using the immunizing peptide

  • Testing cross-reactivity with recombinant ARPC1A protein to ensure isoform specificity

• Application-specific validation:

  • For immunofluorescence: parallel staining with two different ARPC1B antibodies targeting different epitopes

  • For immunoprecipitation: confirming enrichment of known ARPC1B interacting partners (e.g., Aurora A, γ-tubulin)

  • For functional assays: rescue experiments in ARPC1B-deficient cells

Research shows that ARPC1B-deficient patients often exhibit compensatory upregulation of the ARPC1A isoform , making careful validation even more critical. When conducting knockdown experiments, researchers should verify the specificity of ARPC1B depletion by probing for Arpc2, Arp3, and other controls, as demonstrated in published studies .

What are the optimal sample preparation techniques for different ARPC1B antibody applications?

Different applications require tailored sample preparation approaches to maximize ARPC1B detection while preserving relevant biological information:

When studying ARPC1B's role in immune synapse formation, sample preparation should preserve the delicate actin structures at the cell-cell interface. Research has demonstrated that electron microscopy can effectively visualize the interdigitations at immune synapses, which are absent in ARPC1B-deficient CTLs . For cell cycle-dependent studies, synchronized cell populations using methods like double-thymidine block have been successfully employed to study ARPC1B interactions with Aurora A .

How can researchers distinguish between ARPC1B within the Arp2/3 complex versus its independent functions?

Distinguishing between ARPC1B's roles within and outside the Arp2/3 complex requires specialized experimental approaches:

• Biochemical separation:

  • Sucrose gradient sedimentation has been successfully used to separate ARPC1B into two distinct pools: stand-alone ARPC1B (fractions 6-9) and ARPC1B as part of the Arp2/3 complex (fractions 12-15)

  • Size-exclusion chromatography can similarly separate protein complexes based on molecular weight

• Co-immunoprecipitation strategies:

  • Immunoprecipitation with anti-ARPC1B antibodies followed by blotting for other Arp2/3 components identifies ARPC1B within the complex

  • Reciprocal immunoprecipitation using antibodies against Aurora A or γ-tubulin can pull down ARPC1B independent of the Arp2/3 complex

  • Western blotting for Arp3 can serve as a control to confirm complex integrity or separation

• Functional discrimination:

  • In vitro kinase assays with purified components demonstrated that ARPC1B enhances Aurora A kinase activity, while neither Arp3 nor ARPC1A showed this ability

  • While the purified Arp2/3 complex enhanced Aurora A activity, this was attributed to the presence of ARPC1B within the complex

Research has demonstrated that γ-tubulin co-immunoprecipitates with endogenous ARPC1B and Aurora A but not with Arp3 from cells arrested in metaphase, supporting the independent function of ARPC1B at centrosomes . Furthermore, sucrose gradient experiments showed that γ-tubulin co-existed with ARPC1B in both the ARPC1B-only fractions (6-9) and the Arp2/3 complex fractions (12-15), providing additional biochemical evidence for ARPC1B's dual functionality .

What are common issues in immunofluorescence with ARPC1B antibodies and how can they be resolved?

Immunofluorescence with ARPC1B antibodies can encounter several challenges, particularly when studying its dynamic localization in immune cells or during cell division. Common issues and their solutions include:

• High background signal:

  • Increase blocking duration (use 5-10% serum from the same species as the secondary antibody)

  • Optimize antibody dilution (typically 1:100-1:500 for primary antibodies)

  • Include 0.1-0.3% Triton X-100 in the blocking buffer to reduce non-specific binding

• Poor co-localization with actin structures:

  • Ensure appropriate fixation (4% paraformaldehyde preserves actin structures better than methanol)

  • Use gentle permeabilization (0.1% Triton X-100 for 5 minutes)

  • Consider using ARPC1B antibodies targeting different epitopes, as some may be masked in certain conformations

• Difficulty detecting centrosomal ARPC1B:

  • Use cell synchronization (e.g., double-thymidine block) to enrich for mitotic cells

  • Include co-staining with centrosomal markers (e.g., γ-tubulin or centrin)

  • Apply image deconvolution to improve resolution of centrosomal structures

• Weak signal at the immune synapse:

  • Fix cells promptly after synapse formation to capture transient interactions

  • Use conjugated ARPC1B antibodies (e.g., FITC-conjugated) for improved signal

  • Consider tyramide signal amplification for enhanced sensitivity

Research has employed superresolution microscopy for visualizing ARPC1B and actin structures in CTLs, revealing thick lamellipodia at the leading edge in cells migrating on ICAM-1–coated surfaces, appearing as a peripheral ring of dense, branched actin in cells spread on anti-CD3–coated surfaces . This approach provides superior resolution for capturing the intricate cytoskeletal arrangements dependent on ARPC1B.

How can researchers optimize ARPC1B detection in Western blotting?

Western blotting for ARPC1B requires careful optimization to ensure specific detection, particularly when analyzing patient samples or knockdown efficiency. Key considerations include:

• Sample preparation optimization:

  • Include protease inhibitors to prevent degradation

  • For phosphorylation studies, add phosphatase inhibitors

  • Load appropriate protein amounts (typically 20-50 μg of total protein)

• Gel electrophoresis and transfer considerations:

  • Use 10-12% polyacrylamide gels for optimal resolution around 41 kDa

  • Consider wet transfer for more efficient transfer of proteins

  • Use PVDF membranes for higher protein binding capacity

• Antibody optimization:

  • Test multiple antibody dilutions (typically starting at 1:1000)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Use 5% non-fat dry milk in TBST for blocking and antibody dilution

• Verification strategies:

  • Include positive controls (e.g., recombinant ARPC1B)

  • Run ARPC1B-deficient samples as negative controls

  • Probe for loading controls (e.g., β-actin or GAPDH) and specificity controls (e.g., Arpc2 or Arp3)

Western blotting has been effectively used to verify ARPC1B deficiency in patient-derived CTLs, simultaneously demonstrating compensatory upregulation of the ARPC1A isoform . When performing knockdown experiments, including controls for other Arp2/3 complex components (Arpc2, Arp3) and relevant proteins (Aurora A) can verify the specificity of ARPC1B depletion .

What strategies can improve the detection of ARPC1B-protein interactions?

Detecting ARPC1B interactions with partner proteins requires techniques that preserve complex integrity while providing sufficient specificity. Optimal approaches include:

• Co-immunoprecipitation optimization:

  • Use gentle lysis buffers (e.g., NP-40 or digitonin-based)

  • Include stabilizing agents like 10% glycerol in buffers

  • Consider crosslinking approaches for transient interactions

  • Perform reciprocal immunoprecipitations (IP with anti-ARPC1B and anti-partner protein)

• Proximity-based interaction detection:

  • Proximity ligation assay (PLA) for in situ detection of protein-protein interactions

  • FRET or BRET approaches for live-cell interaction studies

  • BioID or APEX2 proximity labeling for identifying novel interaction partners

• Cell cycle-specific interaction analysis:

  • Synchronize cells using double-thymidine block to enrich for specific cell cycle phases

  • Release synchronized cells at different time points to track temporal dynamics of interactions

  • Include cell cycle markers to correlate interactions with specific phases

Research has successfully employed reciprocal immunoprecipitation using anti-Aurora A or anti-ARPC1B antibodies to demonstrate their cell cycle-dependent interaction at the 8-hour time point after release from double-thymidine block . This approach revealed that endogenous Aurora A and ARPC1B interact specifically during mitosis, highlighting the importance of considering cell cycle stage when studying ARPC1B interactions.

How can ARPC1B antibodies contribute to understanding immunological disorders?

ARPC1B antibodies represent powerful tools for investigating the molecular basis of immunological disorders, particularly those involving cytoskeletal dysfunction in immune cells. Strategic applications include:

• Diagnostic applications:

  • Western blotting with ARPC1B antibodies can identify patients with ARPC1B deficiency

  • Parallel probing for ARPC1A can reveal compensatory upregulation, a characteristic feature of ARPC1B deficiency

  • Flow cytometry with ARPC1B antibodies can quantify protein levels in specific immune cell populations

• Mechanistic investigations:

  • Immunofluorescence can reveal defects in immune synapse formation and actin reorganization

  • Co-immunoprecipitation can identify altered protein-protein interactions in disease states

  • Live cell imaging with ARPC1B antibodies can track dynamic processes affected in patients

• Therapeutic development:

  • Antibodies can be used to evaluate the effectiveness of gene therapy approaches

  • Screening for compounds that modify ARPC1B function or bypass ARPC1B deficiency

  • Monitoring restored protein expression following therapeutic interventions

Research has demonstrated that ARPC1B deficiency leads to multiple cellular defects, including impaired lamellipodia formation, defective immune synapse formation, and compromised receptor recycling via the retromer and WASH complexes . These findings provide molecular explanations for the clinical symptoms observed in patients, including decreased CD8+ T cell counts and increased susceptibility to viral infections. ARPC1B antibodies are essential tools for dissecting these pathways and developing targeted interventions.

What are emerging techniques that enhance ARPC1B research?

The field of ARPC1B research continues to evolve with the integration of cutting-edge techniques that provide unprecedented insights into protein function and dynamics:

• Advanced imaging approaches:

  • Super-resolution microscopy (STORM, PALM, SIM) for nanoscale visualization of ARPC1B localization

  • Lattice light-sheet microscopy for 4D imaging of ARPC1B dynamics in living cells

  • Expansion microscopy for enhanced spatial resolution of cytoskeletal structures

• Genome editing technologies:

  • CRISPR-Cas9 for generating precise ARPC1B knockout or knockin models

  • Base editing for introducing patient-specific ARPC1B mutations

  • Prime editing for correcting pathogenic ARPC1B variants

• Proteomics and interactomics:

  • BioID or APEX2 proximity labeling to map the ARPC1B interactome

  • Crosslinking mass spectrometry (XL-MS) to identify direct binding interfaces

  • Thermal proteome profiling to assess ARPC1B stability and complex formation

• Single-cell approaches:

  • Single-cell RNA-seq combined with protein detection (CITE-seq) to correlate ARPC1B expression with cellular phenotypes

  • Single-cell western blotting for heterogeneity analysis of ARPC1B expression

  • Imaging mass cytometry for multiplexed protein detection in tissues

These emerging techniques promise to expand our understanding of ARPC1B beyond its structural role in the Arp2/3 complex to encompass its independent functions in cellular processes like mitosis, receptor trafficking, and immune synapse formation. The integration of multimodal approaches will be particularly valuable for translating basic ARPC1B research into clinical applications for immunodeficiency disorders.