ACTR1A Antibody

What is ACTR1A and what cellular functions does it participate in?

ACTR1A (also known as ARP1, Arp1A, CTRN1, Alpha-centractin) is a 42.6 kD subunit of dynactin, a macromolecular complex consisting of 10-11 subunits. It serves as the most abundant component in the dynactin complex, present in 8-13 copies per molecule. ACTR1A is approximately 60% identical at the amino acid level to conventional actin and forms part of the filament around which the dynactin complex is built .

Functionally, ACTR1A is involved in:

• ER-to-Golgi transport

• Centripetal movement of lysosomes and endosomes

• Spindle formation

• Chromosome movement

• Nuclear positioning

• Axonogenesis

As part of the dynactin complex, it activates the molecular motor dynein for ultra-processive transport along microtubules, playing a crucial role in intracellular organization and transport .

How can I determine which ACTR1A antibody is suitable for my specific research application?

Selecting the appropriate ACTR1A antibody requires careful consideration of several experimental factors:

For Western blot analysis, antibodies like ab203833 have been validated at 1/1000 dilution against human cell lines including Jurkat, MOLT-4, and HeLa cells with a predicted band size of 42 kDa . For immunohistochemistry, antibodies have been tested on mouse cerebellum tissue with recommended antigen retrieval using TE buffer at pH 9.0 or alternatively citrate buffer at pH 6.0 .

Always perform preliminary validation experiments in your specific experimental system, as antibody performance can vary significantly across different applications and sample types .

How is ACTR1A involved in immune signaling pathways, and what methodological approaches can be used to study this interaction?

Recent proteomics studies have identified ACTR1A as a novel regulator of Toll-like receptor 2 (TLR2) signaling. Using co-immunoprecipitation-based cross-linking proteomics with different cross-linkers of varying spacer chain lengths, researchers identified ACTR1A as a potential interactor of TLR2 .

Methodological approach:

• Cross-linking proteomics: Utilizing chemical cross-linkers to fix protein complexes followed by co-immunoprecipitation and mass spectrometry.

• RNA interference validation: Functional studies using siRNA to silence ACTR1A expression confirmed its requirement for TLR2 signaling leading to pro-inflammatory cytokine induction.

• Gene expression analysis: qPCR measurement of TNFα, IL-6, and IL-8 expression in cells with and without ACTR1A silencing.

Interestingly, in HEK293 cells, ACTR1A knockdown significantly affected TLR2-dependent cytokine production. While TNFα response to Pam3CSK4 (P3C, a TLR2 agonist) was not modified by silencing ACTR1A, the TNFα response to combined P3C-statin treatment was significantly inhibited, suggesting statins augment TLR2-dependent TNFα through an ACTR1A-dependent mechanism. For IL-8, both P3C induction and P3C-statin treatment responses were significantly reduced by ACTR1A silencing, indicating ACTR1A requirement for TLR2 responses .

What are the most effective methods for visualizing ACTR1A in cellular contexts, and how can co-localization with binding partners be optimized?

For optimal visualization of ACTR1A and its potential co-localization with binding partners:

Immunofluorescence protocol optimization:

• Cell preparation: Grow cells on acid-treated glass slides (1M HCl-treated)

• Fixation: Use chilled methanol for 5 minutes at room temperature

• Permeabilization: 0.1% Triton X-100 in 1× PBS for 10 minutes

• Blocking: Bovine serum albumin and glycine in 1× PBS for 30 minutes at room temperature in darkness

• Primary antibody incubation: Anti-ACTR1A antibody (e.g., ab11009, Abcam) at 4°C overnight in darkness

• Secondary antibody incubation: Fluorescently labeled secondary antibody (e.g., goat anti-rabbit IgG H&L, Alexa Fluor 488) for 2 hours at room temperature in darkness

• Nuclear counterstaining: DAPI for nuclear visualization

• Imaging: Confocal microscopy with appropriate laser settings

For co-localization studies with potential binding partners such as TLR2, parallel staining with both anti-ACTR1A and anti-TLR2 antibodies can be performed using secondary antibodies with spectrally distinct fluorophores. Adjust antibody concentrations and incubation times based on preliminary optimization experiments to maximize signal-to-noise ratio .

What is the current understanding of ACTR1A's role in pathological conditions, and how are ACTR1A antibodies contributing to this research?

Research indicates potential roles for ACTR1A in several pathological contexts:

• Cancer research: ACTR1A antibodies have been used in studying cancer contexts, with immunohistochemical analysis of human lung cancer tissue showing positive staining patterns . Additionally, genetic testing for ACTR1A is associated with medulloblastoma, suggesting a potential role in cancer development or progression .

• Inflammatory pathways: Through its interaction with TLR2, ACTR1A may play a role in inflammatory conditions. Knockdown studies have demonstrated its influence on pro-inflammatory cytokine production, with potential implications for inflammatory diseases .

• Neurodegenerative diseases: As part of the dynactin complex involved in vesicular transport, ACTR1A may have relevance in neurodegenerative conditions. Studies have examined its potential interaction with leucine-rich repeat kinase 2 (LRRK2), which is associated with Parkinson's disease .

Researchers utilizing ACTR1A antibodies in these contexts should consider:

• Using multiple antibody clones to validate findings

• Combining with genetic approaches (siRNA, CRISPR) for functional validation

• Correlating protein expression with clinical outcomes in patient samples

• Employing tissue microarrays for high-throughput screening across multiple disease states

What are the common technical challenges in Western blot detection of ACTR1A, and how can these be addressed?

Several challenges may arise when detecting ACTR1A via Western blot:

Challenge 1: Multiple band detection
ACTR1A antibodies may detect multiple bands due to:

• Predicted band sizes for ACTR1A include 42 kDa (observed), but also potential bands at 53 kDa and 122 kDa

• Cross-reactivity with related proteins like ACTR1B (Beta-centractin)

• Post-translational modifications

Solution: Validate specificity using positive controls like Jurkat, MOLT-4, or HeLa cell lysates where a clear 42 kDa band should be observed. Consider using knockout/knockdown samples as negative controls to confirm band identity .

Challenge 2: Optimal blocking conditions
Solution: Use 5% non-fat dry milk in TBST as blocking/dilution buffer, which has been validated in multiple published protocols .

Challenge 3: Protein degradation
Solution: Add protease inhibitors to lysates and maintain cold chain during sample preparation. For storage, aliquot samples with 50% glycerol and store at -20°C, avoiding repeated freeze/thaw cycles .

Optimized Western blot protocol for ACTR1A detection:

• Sample preparation: Whole cell lysates at 10-30 μg per lane

• SDS-PAGE: 10% gel for optimal separation

• Transfer: Standard wet transfer to PVDF membrane

• Blocking: 5% non-fat dry milk in TBST

• Primary antibody: Anti-ACTR1A at 1:1000 dilution in blocking buffer, overnight at 4°C

• Secondary antibody: HRP-conjugated at 1:8000 dilution, room temperature for 1 hour

• Development: ECL detection system with appropriate exposure time (typically 3 minutes)

How can researchers effectively validate ACTR1A antibody specificity to ensure reliable experimental results?

Comprehensive validation of ACTR1A antibodies is essential for experimental rigor:

• Multiple detection techniques:

  • Compare results across Western blot, immunohistochemistry, and immunofluorescence

  • Each technique should show consistent ACTR1A detection patterns

• Genetic validation approaches:

  • siRNA/shRNA knockdown: Verify reduction in antibody signal correlates with ACTR1A silencing

  • CRISPR knockout: Use as negative control to confirm antibody specificity

  • Overexpression systems: Test if signal increases proportionally with increased ACTR1A expression

• Cross-antibody validation:

  • Use multiple antibodies targeting different ACTR1A epitopes

  • Compare monoclonal (e.g., EPR16968(B)) and polyclonal (e.g., 10357-1-AP) antibodies

  • Consistent results across different antibodies increases confidence in specificity

• Species reactivity testing:

  • Verify predicted cross-reactivity with human, mouse, and rat samples

  • Use species-specific positive controls to confirm reactivity claims

  • Remember that species cross-reactivity is based on epitope conservation

• Application-specific controls:

  • For Western blot: Include molecular weight markers and verify expected band size (42-45 kDa)

  • For IHC: Include tissue with known ACTR1A expression (e.g., brain tissue)

  • For IP: Perform reverse IP and confirm co-precipitated proteins

What are the best approaches for optimizing immunoprecipitation experiments using ACTR1A antibodies, particularly for identifying novel interaction partners?

For researchers investigating ACTR1A protein-protein interactions:

Standard IP protocol optimization:

• Lysis buffer selection: Use buffers that maintain native protein conformations while effectively disrupting membranes (e.g., RIPA or NP-40-based buffers with protease inhibitors)

• Antibody selection: Recombinant monoclonal antibodies like EPR16968(B) have been validated for IP applications with human samples

• Antibody-to-lysate ratio: Typically 2-5 μg antibody per 500 μg total protein

• Pre-clearing: Remove non-specific binding proteins using protein A/G beads before adding the specific antibody

• Incubation conditions: Overnight at 4°C with gentle rotation

• Washing stringency: Balance between removing non-specific interactions and maintaining specific ones

Advanced IP approaches for novel interaction discovery:

• Cross-linking IP: As demonstrated in TLR2-ACTR1A interaction studies, use membrane-permeable cross-linkers of different spacer lengths to stabilize transient or weak interactions before cell lysis

• Proximity-dependent labeling: Consider BioID or APEX2 fusion proteins to identify proteins in close proximity to ACTR1A in living cells

• Mass spectrometry analysis: After IP, analyze samples using LC-MS/MS to identify co-precipitated proteins

Validation of novel interactions:

• Perform reverse IP using antibodies against the potential interacting partner

• Confirm co-localization by immunofluorescence microscopy

• Use functional assays (e.g., siRNA knockdown) to verify biological relevance of interaction

• Consider in vitro binding assays with purified proteins to confirm direct interaction

How can ACTR1A antibodies contribute to understanding the role of this protein in neurodegenerative diseases?

ACTR1A's function in microtubule-based vesicle motility and its association with the centrosome make it particularly relevant to neurodegenerative disease research . Researchers can utilize ACTR1A antibodies in several strategic approaches:

• Comparative expression studies:

  • Analyze ACTR1A expression in post-mortem brain tissues from patients with neurodegenerative diseases versus controls

  • Immunohistochemistry using optimized protocols (1:250-1:1000 dilution) for human brain tissue

  • Western blot analysis of brain region-specific protein extracts

• Co-localization with disease-associated proteins:

  • Examine potential interactions with proteins implicated in neurodegeneration

  • Studies have begun exploring relationships with LRRK2 (Parkinson's disease) using co-immunoprecipitation approaches

  • Immunofluorescence co-localization in neuronal cell models

• Dynamic vesicular transport analysis:

  • Live-cell imaging of fluorescently-tagged vesicles in neuronal models with manipulated ACTR1A expression

  • Immunostaining for vesicular markers alongside ACTR1A in fixed neuronal preparations

  • Analysis of vesicular transport defects in disease models and correlation with ACTR1A localization/function

• Therapeutic target exploration:

  • Screening compounds that may modulate ACTR1A function or expression

  • Developing antibody-based approaches to monitor treatment effects on dynactin complex integrity

  • Exploring potential correlation between ACTR1A activity and disease progression

Researchers should note that ACTR1A antibodies have been successfully employed in human fetal brain tissue analysis, suggesting utility for developmental neurobiology studies in addition to neurodegeneration research .

What considerations should researchers take into account when designing ACTR1A knockdown or knockout experiments, and how can antibodies validate these approaches?

When manipulating ACTR1A expression for functional studies:

Experimental design considerations:

• Selection of knockdown approach:

  • siRNA: Effective for transient knockdown in most cell types

  • shRNA: For stable knockdown in long-term experiments

  • CRISPR-Cas9: For complete knockout studies, noting that complete loss may be lethal in some cell types

• Potential compensatory mechanisms:

  • Consider potential upregulation of related proteins (e.g., ACTR1B/Beta-centractin)

  • Monitor dynactin complex integrity and function

  • Evaluate phenotypic effects at multiple timepoints after knockdown/knockout

• Cell type-specific effects:

  • Different cell types may exhibit varying sensitivity to ACTR1A reduction

  • Neuronal cells, with extensive vesicular transport requirements, may be particularly affected

  • Cancer cell lines (e.g., Jurkat, MOLT-4, HeLa) have validated ACTR1A expression and can serve as model systems

Antibody-based validation strategies:

• Confirmation of knockdown/knockout efficiency:

  • Western blot with quantification (1:1000-1:8000 dilution range)

  • Immunofluorescence for single-cell level verification

  • Flow cytometry for population-level analysis

• Functional readouts:

  • As shown in TLR2 signaling studies, monitor downstream effects such as cytokine expression

  • Use immunoassays to measure TNFα, IL-6, and IL-8 production in response to stimuli

  • Compare phenotypic effects with published findings to ensure consistency

• Rescue experiments:

  • Re-express ACTR1A (possibly with tag for discrimination from endogenous protein)

  • Verify expression of rescue construct using antibodies

  • Demonstrate restoration of normal function to confirm phenotype specificity

The successful application of ACTR1A knockdown approaches has been demonstrated in HEK293 cells, where siRNA effectively reduced ACTR1A expression and altered cytokine responses to TLR2 stimulation, providing a validated experimental framework that researchers can adapt .

How might advances in antibody technology enhance ACTR1A research, and what new applications are on the horizon?

Emerging antibody technologies offer significant potential for advancing ACTR1A research:

• Single-domain antibodies and nanobodies:

  • Smaller size allows access to epitopes inaccessible to conventional antibodies

  • Better penetration in tissue sections and living cells

  • Potential for intracellular expression to track ACTR1A in real-time

• Multicolor imaging applications:

  • Directly conjugated antibodies with spectrally distinct fluorophores

  • Multiplexed imaging of ACTR1A with interaction partners

  • Super-resolution microscopy techniques to visualize ACTR1A within dynactin complex structure

• Proximity labeling approaches:

  • Antibody-enzyme fusion constructs (e.g., HRP or APEX2 conjugates)

  • Spatially restricted labeling of ACTR1A interaction partners

  • Temporal dynamics of ACTR1A-containing complexes

• Therapeutic applications:

  • Development of antibodies or antibody derivatives that modulate ACTR1A function

  • Potential applications in cancer, given ACTR1A's association with medulloblastoma

  • Targeted delivery of therapeutic payloads to cells with aberrant ACTR1A expression

• Combination with emerging technologies:

  • Integration with spatial transcriptomics to correlate ACTR1A protein localization with gene expression patterns

  • Cryo-electron microscopy for structural studies of ACTR1A within the dynactin complex

  • AI-assisted image analysis for quantitative assessment of ACTR1A distribution patterns

As research continues, the development of more specific, sensitive, and versatile ACTR1A antibodies will enable increasingly sophisticated investigations into this protein's diverse cellular functions and potential roles in disease pathogenesis.

What is the relationship between ACTR1A and ACTR1B antibodies, and how can researchers ensure specificity when studying these closely related proteins?

ACTR1A and ACTR1B (Beta-centractin) are structurally related proteins that can present challenges for antibody-based studies:

Structural and functional relationship:

• Both are actin-related proteins with roles in the dynactin complex

• ACTR1B (Beta-centractin) is also known as Actin-related protein 1B or ARP1B

• They share significant sequence homology, making antibody cross-reactivity a concern

Specificity challenges:

• Epitope selection: Some antibodies like EPR16968(B) recognize both ACTR1A and ACTR1B, which may be intentional for studying the dynactin complex collectively, but problematic for protein-specific studies

• Similar molecular weights: Both have observed molecular weights around 42-45 kDa, making them difficult to distinguish by size alone on Western blots

• Co-expression patterns: Often expressed in the same tissues and cell types, complicating interpretation of staining patterns

Strategies for ensuring specificity:

• Antibody selection:

  • Choose antibodies raised against unique regions not conserved between ACTR1A and ACTR1B

  • Verify epitope information from manufacturers

  • When using antibodies recognizing both proteins (like EPR16968(B)), be clear about this limitation in experimental interpretation

• Validation approaches:

  • Use recombinant proteins of each type as positive controls

  • Employ genetic knockdown/knockout of each protein individually

  • Peptide competition assays with peptides specific to each protein

• Complementary techniques:

  • RT-qPCR to distinguish mRNA expression of each gene

  • Mass spectrometry to identify peptides unique to each protein

  • Co-immunoprecipitation with protein-specific binding partners

• Experimental design considerations:

  • Include appropriate controls in every experiment

  • Consider using multiple antibodies targeting different epitopes

  • Be transparent about potential cross-reactivity in research publications