ACTN1 Monoclonal Antibody

What is ACTN1 and what are its primary functions in cellular biology?

ACTN1 (Alpha-actinin-1) is an F-actin cross-linking protein that anchors actin to various intracellular structures. It functions as a key cytoskeletal protein involved in multiple cellular processes. ACTN1 determines the motility of keratinocytes by regulating the organization of the actin cytoskeleton, focal adhesion, and hemidesmosome protein complexes, thereby modulating cell speed, lamellipodial dynamics, and directed migration . In immune contexts, ACTN1's association with IGSF8 regulates immune synapse formation and is required for efficient T-cell activation . The protein is involved in multiple cellular pathways including adherens junction formation, focal adhesion, leukocyte transendothelial migration, regulation of actin cytoskeleton, and tight junction assembly . Recent research has also revealed its role in cancer progression, particularly in hepatocellular carcinoma where it acts as a tumor promoter by suppressing Hippo signaling via physical interaction with MOB1 .

What applications are ACTN1 monoclonal antibodies suitable for?

ACTN1 monoclonal antibodies have demonstrated utility across multiple experimental applications in molecular and cellular biology research. Based on validated data, these antibodies are suitable for:

The AT1D10 clone (AM50637PU-N) shows broad application versatility across multiple techniques, while some clones like ab18061 have been particularly validated for immunocytochemistry and immunofluorescence applications . When selecting an antibody for a specific application, researchers should consider the validated applications indicated for each clone to ensure optimal experimental results.

What are the recommended protocols for immunofluorescence staining using ACTN1 antibodies?

For optimal immunofluorescence staining of ACTN1, researchers should follow this validated protocol:

• Cell preparation: Seed cells at appropriate density in suitable chamber slides or coverslips (e.g., 12-well U-Chamber) .

• Fixation: Fix cells with either 4% paraformaldehyde for 15 minutes at room temperature or 100% methanol for 5 minutes . The fixative choice depends on the epitope accessibility and experimental goals.

• Permeabilization: If using paraformaldehyde fixation, permeabilize with 0.05% (v/v) Triton X-100 for 1 minute at room temperature .

• Blocking: Block with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1 hour at room temperature to reduce non-specific binding .

• Primary antibody incubation: Incubate with anti-ACTN1 antibody at the recommended dilution (typically 1:100 for research-grade antibodies like ab50599 or 5 μg/mL for ab18061 ) overnight at 4°C.

• Secondary antibody incubation: After washing, incubate with appropriate fluorophore-conjugated secondary antibody (e.g., Alexa Fluor 594-conjugated anti-rabbit or Fluor 488-conjugated anti-mouse) .

• Nuclear counterstaining: Stain nuclei with DAPI.

• Mounting and imaging: Mount slides and capture images using confocal microscopy .

This protocol has been validated for detecting endogenous ACTN1 in various cell types, including ioSkeletal Myocytes derived from human iPSCs and hepatocellular carcinoma cell lines such as MHCC-97H .

How does ACTN1 expression correlate with cancer progression and patient outcomes?

ACTN1 expression shows significant correlation with cancer progression and patient outcomes, particularly in hepatocellular carcinoma (HCC). Comprehensive immunohistochemical analysis of HCC tissue microarrays (n=157) has revealed:

These findings establish ACTN1 as both a potential prognostic marker and a therapeutic target for HCC, with expression analysis using validated monoclonal antibodies serving as a valuable clinical research tool.

What molecular mechanisms underlie ACTN1's role in cancer progression?

ACTN1 promotes cancer progression through several interconnected molecular mechanisms:

• Hippo pathway inhibition: ACTN1 suppresses the Hippo signaling pathway, a critical tumor suppressor pathway. Mechanistically, ACTN1 competitively interacts with MOB1, which decreases the phosphorylation of LATS1 and YAP (key Hippo pathway components) .

• YAP activation: By inhibiting the Hippo pathway, ACTN1 promotes YAP activity, which drives oncogenic gene expression. This effect can be abrogated by pharmacological inhibition of YAP with verteporfin or super-TDU .

• Rho GTPase modulation: Knockdown of ACTN1 decreases Rho GTPases activities, which are important regulators of cytoskeletal dynamics and cell mobility .

• Cell proliferation regulation: In vitro loss-of-function studies demonstrate that ACTN1 knockdown suppresses HCC cell proliferation .

• In vivo tumor growth promotion: Both subcutaneous xenograft models and intrahepatic transplantation models confirm that ACTN1 contributes significantly to tumor growth of HCC .

These mechanisms highlight the multifaceted role of ACTN1 in cancer biology and suggest that monoclonal antibodies against ACTN1 are valuable tools for investigating these pathways in experimental models.

How can researchers effectively validate ACTN1 antibody specificity?

Validating ACTN1 antibody specificity is critical for reliable experimental results. A comprehensive validation approach should include:

• Genetic knockdown/knockout controls:

  • Compare staining patterns between wild-type cells and those with ACTN1 knockdown/knockout

  • Loss of signal in ACTN1-depleted samples confirms antibody specificity

• Multi-technique validation:

  • Validate expression using complementary methods (e.g., western blot, immunofluorescence, and flow cytometry)

  • Consistent results across techniques strengthen confidence in antibody specificity

• Subcellular localization assessment:

  • Confirm expected cytoplasmic distribution of ACTN1

  • Co-localization with known interacting partners (e.g., actin filaments or MOB1)

• Positive and negative tissue controls:

  • Use tissues with known high ACTN1 expression (e.g., HCC tissues) as positive controls

  • Use matched non-cancerous tissues as negative/low expression controls

• Recombinant protein competition:

  • Pre-incubate antibody with recombinant ACTN1 protein (such as the immunogen fragment used to generate the antibody)

  • Specific binding should be competitively inhibited

• Cross-reactivity testing:

  • Test against other ACTN family members (ACTN2, ACTN3, ACTN4) to ensure isoform specificity

  • Important as these proteins share structural similarity but have distinct functions

Implementing these validation approaches ensures that observed signals genuinely represent ACTN1 rather than non-specific or off-target binding.

What are the critical parameters for western blot optimization when using ACTN1 antibodies?

Optimizing western blot procedures for ACTN1 detection requires attention to several critical parameters:

• Sample preparation:

  • Include protease inhibitors to prevent ACTN1 degradation

  • Optimize lysis buffer composition based on subcellular localization (cytoplasmic vs. cytoskeletal fraction)

  • For cytoskeletal-associated ACTN1, use buffers containing non-ionic detergents that preserve protein-protein interactions

• Protein loading and separation:

  • ACTN1 has a molecular weight of approximately 100-103 kDa

  • Use 8-10% acrylamide gels for optimal resolution

  • Load 20-50 μg of total protein per lane for cell lysates

• Transfer conditions:

  • Wet transfer is recommended for high molecular weight proteins like ACTN1

  • Extended transfer time (90-120 minutes) at controlled temperature may improve efficiency

• Blocking and antibody incubation:

  • Block with 5% non-fat milk or BSA in TBST

  • Use the recommended antibody dilution (typically 1:1000 for western blot)

  • For clone AT1D10, overnight incubation at 4°C yields optimal results

• Controls and normalization:

  • Include positive control lysates from cells known to express ACTN1

  • Use appropriate housekeeping proteins (β-actin, GAPDH) for normalization

  • Consider including ACTN1 knockdown/knockout samples as negative controls

• Signal detection:

  • Both chemiluminescence and fluorescence-based detection systems are suitable

  • Optimize exposure time to prevent signal saturation for accurate quantification

These parameters should be systematically optimized to achieve reproducible and quantifiable detection of ACTN1 in experimental samples.

How should researchers design co-immunoprecipitation experiments to study ACTN1 interactions?

Co-immunoprecipitation (Co-IP) experiments are valuable for studying ACTN1 protein interactions, particularly with partners like MOB1 . A well-designed Co-IP protocol includes:

• Experimental planning:

  • Define clear hypotheses about specific ACTN1 interaction partners

  • Consider epitope accessibility in protein complexes

  • Plan appropriate controls (IgG control, input samples, reciprocal Co-IP)

• Lysis conditions:

  • Use gentle lysis buffers that preserve protein-protein interactions

  • Recommended buffer: 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, with protease and phosphatase inhibitors

  • Avoid harsh detergents like SDS that disrupt protein interactions

• Pre-clearing lysates:

  • Pre-clear with protein A/G beads to reduce non-specific binding

  • Incubate lysate with beads for 1 hour at 4°C before the actual IP

• Antibody selection:

  • Choose antibodies with validated IP applications

  • For ACTN1-MOB1 interaction studies, both anti-ACTN1 and anti-MOB1 antibodies should be used in reciprocal Co-IPs

• Immunoprecipitation procedure:

  • Incubate pre-cleared lysate with 2-5 μg antibody overnight at 4°C

  • Add protein A/G beads and incubate for 2-4 hours at 4°C

  • Wash extensively (4-5 times) with lysis buffer to remove non-specific binding

• Elution and analysis:

  • Elute with SDS sample buffer at 95°C for 5 minutes

  • Analyze by western blotting using antibodies against both the bait (ACTN1) and suspected interacting proteins

• Validation approaches:

  • Perform reciprocal Co-IP (e.g., IP with anti-MOB1 and blot for ACTN1)

  • Include competitive peptide controls to confirm specificity

  • Correlate Co-IP results with other interaction methods (e.g., immunofluorescence co-localization)

Following this approach has successfully demonstrated the physical interaction between ACTN1 and MOB1, a key finding in understanding ACTN1's role in Hippo pathway regulation .

What methodological considerations are important for studying subcellular localization of ACTN1?

Studying the subcellular localization of ACTN1 requires careful methodological considerations:

Following these methodological considerations has enabled researchers to effectively visualize ACTN1 in various contexts, including ioSkeletal Myocytes (human iPSC-derived) and hepatocellular carcinoma cell lines .

What are common issues in ACTN1 immunodetection and how can they be addressed?

Researchers may encounter several challenges when detecting ACTN1. Here are common issues and their solutions:

• High background in immunofluorescence:

  • Cause: Insufficient blocking or excessive antibody concentration

  • Solution: Increase blocking time (≥1 hour), use 1% BSA/10% normal goat serum/0.3M glycine in PBS-Tween , and optimize antibody dilutions (typically 1:100-1:200 for primary antibodies)

• Weak or absent signal:

  • Cause: Epitope masking, inadequate permeabilization, or protein degradation

  • Solution: Test different fixation methods (compare methanol vs. paraformaldehyde), increase permeabilization time, and ensure fresh samples with protease inhibitors

• Non-specific bands in western blot:

  • Cause: Cross-reactivity with other actinin family members or non-specific binding

  • Solution: Increase washing stringency, optimize antibody concentration, and validate with ACTN1 knockdown controls

• Inconsistent immunoprecipitation results:

  • Cause: Harsh lysis conditions disrupting protein complexes or inefficient antibody binding

  • Solution: Use gentler lysis buffers, pre-clear lysates thoroughly, and select antibodies validated for immunoprecipitation

• Variable staining patterns across cell types:

  • Cause: Different ACTN1 expression levels or subcellular distributions

  • Solution: Adjust exposure settings, optimize fixation conditions for each cell type, and correlate with western blot quantification

• Poor reproducibility:

  • Cause: Lot-to-lot antibody variation or inconsistent experimental conditions

  • Solution: Document antibody lot numbers, standardize protocols with detailed SOPs, and include positive control samples in each experiment

By systematically addressing these issues, researchers can achieve consistent and reliable detection of ACTN1 across various experimental applications.

How can ACTN1 monoclonal antibodies be used to investigate cancer biomarkers and therapeutic targets?

ACTN1 monoclonal antibodies offer valuable tools for investigating cancer biomarkers and therapeutic targets:

These applications demonstrate how ACTN1 monoclonal antibodies serve as vital tools in translational cancer research, bridging basic molecular mechanisms to clinical applications.

What approaches can be used to study post-translational modifications of ACTN1?

Investigating post-translational modifications (PTMs) of ACTN1 requires specialized approaches:

• Phosphorylation analysis:

  • Use phospho-specific antibodies targeting known ACTN1 phosphorylation sites

  • Combine with lambda phosphatase treatment as a control

  • Employ mass spectrometry to identify novel phosphorylation sites

  • Correlate phosphorylation status with functional changes in cytoskeletal organization

• Ubiquitination detection:

  • Immunoprecipitate ACTN1 under denaturing conditions to maintain ubiquitin linkages

  • Probe with anti-ubiquitin antibodies in western blot

  • Use proteasome inhibitors (MG132) to enhance detection of ubiquitinated species

  • Investigate how ubiquitination affects ACTN1 stability and function

• Acetylation assessment:

  • Immunoprecipitate ACTN1 and probe with anti-acetyl-lysine antibodies

  • Treat cells with histone deacetylase inhibitors to enhance acetylation

  • Investigate how acetylation affects ACTN1's interaction with binding partners like MOB1

• PTM crosstalk analysis:

  • Study how different modifications influence each other

  • Investigate how PTMs regulate ACTN1's ability to suppress Hippo signaling

  • Examine how treatment with kinase inhibitors affects other modifications

• Functional consequence investigation:

  • Generate site-specific mutants (phospho-mimetic or phospho-resistant)

  • Compare cellular localization of modified vs. unmodified ACTN1

  • Assess impact on protein-protein interactions, particularly with MOB1 and Hippo pathway components

• Context-dependent modification:

  • Compare modification patterns across different cell types

  • Investigate changes in modification status during cancer progression

  • Examine how microenvironmental factors influence ACTN1 modifications

These approaches provide a comprehensive framework for understanding how post-translational modifications regulate ACTN1 function in both normal physiology and disease states.

What are emerging research areas involving ACTN1 monoclonal antibodies?

Several cutting-edge research areas are emerging for ACTN1 monoclonal antibodies:

• Single-cell analysis: Integration of ACTN1 antibodies into single-cell proteomics workflows to examine expression heterogeneity within tumors and correlate with cellular phenotypes.

• Liquid biopsy development: Investigating ACTN1 as a potential circulating biomarker in cancer patients, using highly sensitive detection methods with monoclonal antibodies.

• Mechanobiology: Exploring ACTN1's role in mechanotransduction and how forces affect its interactions with binding partners like MOB1 .

• Therapeutic antibody development: Engineering anti-ACTN1 antibodies that can modulate its function, particularly its interaction with the Hippo pathway, for potential cancer therapy.

• Multiplexed imaging: Incorporating ACTN1 antibodies into highly multiplexed imaging platforms (CyTOF, CODEX, etc.) to simultaneously visualize multiple components of ACTN1-associated pathways.

• Spatial transcriptomics correlation: Combining ACTN1 protein detection with spatial transcriptomics to correlate protein expression with gene expression patterns in tissue context.

• Drug screening platforms: Using ACTN1 antibodies to develop high-content screening assays for compounds that modulate ACTN1 function or expression.

These emerging areas highlight the continuing importance of high-quality ACTN1 monoclonal antibodies as tools for advancing our understanding of this protein's diverse roles in health and disease.

How can researchers evaluate and compare different commercial ACTN1 antibodies?

Researchers should employ a systematic approach to evaluate and compare ACTN1 monoclonal antibodies:

• Epitope mapping:

  • Compare antibodies targeting different ACTN1 domains

  • Clone AT1D10 recognizes an epitope within amino acids 1-249

  • Different epitopes may be more accessible in certain applications or experimental conditions

• Performance across applications:

  • Evaluate each antibody across multiple applications using standardized protocols

  • Create a comparison matrix of performance metrics for each application

  • Some antibodies perform better in specific applications (e.g., ab18061 for ICC )

• Species reactivity:

  • Test cross-reactivity with ACTN1 from different species if working with animal models

  • Confirm human reactivity for translational research

• Sensitivity and specificity assessment:

  • Determine detection limits using dilution series of recombinant protein

  • Test against ACTN1 knockdown/knockout samples to confirm specificity

  • Evaluate cross-reactivity with other actinin family members (ACTN2-4)

• Lot-to-lot consistency:

  • Test multiple lots of the same antibody clone

  • Maintain reference samples for comparison across experiments

• Validation documentation:

  • Review manufacturer validation data and publications citing each antibody

  • Consider antibodies with extensive citation records in relevant applications

• Balanced assessment:

  • Create a weighted scoring system based on critical parameters for your research

  • Consider cost-effectiveness for long-term projects

This systematic evaluation approach ensures selection of the most appropriate ACTN1 antibody for specific research applications while maximizing experimental reproducibility and data quality.

What are the key considerations for implementing ACTN1 as a biomarker in clinical research studies?

Implementing ACTN1 as a biomarker in clinical research requires careful consideration of several factors: