ACTN1 Antibody

What is ACTN1 and what cellular functions does it mediate?

ACTN1 (Alpha-actinin-1) is a non-muscle, cytoskeletal alpha actinin isoform that functions as an F-actin cross-linking protein. It anchors actin to various intracellular structures and plays multiple roles in cell biology. In non-muscle cells, ACTN1 is found along microfilament bundles and adherens-type junctions, where it binds actin to the membrane . It has important functions in immune synapse formation and is required for efficient T-cell activation. Association with IGSF8 specifically regulates the immune synapse formation .

How do I select the appropriate ACTN1 antibody for my specific research application?

Selection of the appropriate ACTN1 antibody depends on your experimental needs:

• Application compatibility: Verify antibody validation for your technique (WB, IHC, ICC/IF, IP, FACS)

• Species reactivity: Confirm cross-reactivity with your experimental model (human, mouse, rat)

• Epitope consideration: Choose antibodies targeting different regions for specific isoform detection

• Clone type: Consider monoclonal (e.g., OTI7A4, AT6/172) for consistency or polyclonal for broader epitope recognition

• Validation evidence: Review cited publications and validation data

For immunocytochemistry, antibodies like ab18061 are validated for human samples . For broader applications including IHC-P, WB, and ICC/IF with cross-reactivity to human, mouse, and rat, consider antibodies like ab90421 .

What is the optimal protocol for immunofluorescence detection of ACTN1 in fixed cells?

For optimal immunofluorescence detection of ACTN1:

• Fixation: Fix cells with 4% paraformaldehyde for 15 minutes (alternatives: 100% methanol for 5 minutes)

• Permeabilization: Treat with 0.05% (v/v) Triton X-100 for 1 minute at room temperature

• Blocking: Use 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1 hour

• Primary antibody: Incubate with ACTN1 antibody (5 μg/mL for ab18061 or 1:100 for ab50599) for 90 minutes or overnight at 4°C

• Secondary antibody: Apply Alexa Fluor conjugated antibodies (e.g., Alexa Fluor 594-conjugated anti-rabbit or Fluor 488-conjugated anti-mouse)

• Nuclear counterstain: Use DAPI for nuclear visualization

• Imaging: Capture signals using confocal microscopy

For co-localization studies with other proteins (e.g., MOB1), adjust antibody concentrations accordingly and select compatible fluorophores for multiplexing .

What are the critical parameters for successful ACTN1 Western blot analysis?

For reproducible ACTN1 Western blot results:

• Sample preparation:

  • Use RIPA buffer with protease inhibitors

  • Avoid repeated freeze-thaw cycles

• Protein loading:

  • Load 10-20 μg total protein per lane

  • Include positive controls (HepG2, HeLa cells)

• Antibody selection and dilution:

  • Primary: ab90421 at 1:200, ab68194 (EP2527Y) for increased specificity

  • Secondary: IRDye 800CW-conjugated secondary antibodies for enhanced detection

• Optimization considerations:

  • Extended transfer time (>1 hour) for this high molecular weight protein

  • Adequate blocking (5% milk or BSA)

  • Stringent washing to reduce background

• Controls:

  • Include β-actin (Abcam, ab8227, diluted at 1:1000) as loading control

What is the recommended protocol for immunohistochemical detection of ACTN1 in tissue sections?

For reliable ACTN1 immunohistochemistry:

• Tissue preparation:

  • Use 5-μm thick paraffin-embedded sections

  • Deparaffinize with xylene and ethanol

• Antigen retrieval:

  • Heat in 0.1 mol/L citrate buffer (pH 6.0) by microwaving for 15 minutes

  • Cool to room temperature

• Endogenous peroxidase blocking:

  • Incubate in 3% hydrogen peroxide for 20 minutes

• Non-specific binding reduction:

  • Block with 10% BSA

• Antibody application:

  • Primary: ACTN1 antibody (Abcam, ab50599) at 1:200 dilution, 4°C overnight

  • Secondary: HRP-conjugated detection system

• Visualization:

  • Develop with DAB substrate liquid

  • Counterstain with hematoxylin

How should ACTN1 expression be quantified in immunohistochemical studies?

For standardized ACTN1 expression quantification in IHC:

Scoring System Components:

Calculation and Interpretation:

• Calculate total score = staining intensity score × percentage of positive cells score

• Categorize expression levels:

  • Low expression: 1-4

  • Moderate expression: 5-8

  • High expression: 9-12

This standardized approach allows reliable comparison across samples and studies, facilitating consistent interpretation of ACTN1 expression patterns.

What statistical approaches are recommended for analyzing ACTN1 expression data in cancer studies?

For robust statistical analysis of ACTN1 expression in cancer research:

How can ACTN1 antibodies be used to investigate its role in cancer progression?

To investigate ACTN1's role in cancer progression:

• Expression analysis in tissues:

  • Compare ACTN1 levels between tumor and adjacent normal tissues

  • Correlate with clinicopathological features (e.g., studies show ACTN1 is significantly overexpressed in HCC compared to non-cancerous liver tissues)

• Molecular mechanism studies:

  • Examine ACTN1's interaction with signaling pathways (e.g., Hippo pathway)

  • Investigate physical interaction with regulatory proteins (e.g., MOB1)

• Functional assays following ACTN1 manipulation:

  • Assess effects on cell cycle progression, apoptosis, and EMT

  • Evaluate impact on cell proliferation, migration, and invasion using:

    • Cell Counting Kit-8 (CCK8) assays

    • Flow cytometry analysis

    • Transwell assays

    • Wound-healing assays

• In vivo validation:

  • Examine tumor growth in subcutaneous xenograft models

  • Assess metastatic potential using pulmonary metastasis models

Research has demonstrated that ACTN1 protein levels significantly associate with clinical stage and node metastasis, with high expression indicating poor prognosis in multiple cancer types .

What approaches can be used to study ACTN1's interaction with the Hippo signaling pathway?

To investigate ACTN1's interaction with Hippo signaling:

• Protein interaction studies:

  • Perform co-immunoprecipitation to detect physical interactions between ACTN1 and Hippo pathway components (e.g., MOB1)

  • Conduct immunofluorescence analysis to assess co-localization

• Signaling pathway assessment:

  • Following ACTN1 knockdown or overexpression, analyze:

    • Phosphorylated LATS1 (Cell Signaling Technology, #9157, 1:1000)

    • Total LATS1 (Cell Signaling Technology, #3477, 1:1000)

    • Phosphorylated YAP (Cell Signaling Technology, #13008, 1:1000)

    • Total YAP (Cell Signaling Technology, #14074, 1:1000)

• Functional validation:

  • Assess nuclear localization of YAP/TAZ following ACTN1 manipulation

  • Evaluate expression of Hippo pathway target genes

Research has shown that ACTN1 can act as a tumor promoter by suppressing Hippo signaling through physical interaction with MOB1 in hepatocellular carcinoma .

How can I investigate the role of ACTN1 in cytoskeletal dynamics and cell adhesion?

To study ACTN1's function in cytoskeletal dynamics and adhesion:

• Live-cell imaging:

  • Visualize ACTN1 localization during cytoskeletal remodeling using fluorescently-tagged antibodies

  • Monitor redistribution during cell migration and adhesion formation

• Co-localization studies:

  • Examine association with actin filaments and adhesion proteins

  • Use multiple antibodies for simultaneous detection of ACTN1 and binding partners

• Functional manipulation:

  • Perform ACTN1 knockdown and assess effects on:

    • Focal adhesion formation and turnover

    • Cell spreading and attachment dynamics

    • Actin filament organization

• Mechanotransduction analysis:

  • Study ACTN1's role in transmitting mechanical signals from the extracellular matrix

  • Investigate interactions with integrin-linked kinases and downstream signaling

This approach leverages ACTN1's fundamental role as an actin-binding protein involved in anchoring microfilaments to various structures and connecting them to membrane proteins like integrins .

What are common issues encountered with ACTN1 antibodies in immunofluorescence and how can they be resolved?

Common immunofluorescence issues and solutions:

How can I optimize ACTN1 detection in challenging tissue types or conditions?

For optimizing ACTN1 detection in difficult samples:

• Enhanced antigen retrieval:

  • Test multiple buffers (citrate pH 6.0 vs. EDTA pH 9.0)

  • Explore alternative heating methods (microwave vs. pressure cooker)

  • Optimize retrieval time (15-30 minutes)

• Signal amplification strategies:

  • Employ tyramide signal amplification systems

  • Use biotin-streptavidin detection methods

  • Consider polymer-based detection systems

• Antibody optimization:

  • Test multiple ACTN1 antibodies targeting different epitopes

  • Adjust antibody concentration based on tissue type

  • Extend incubation times (overnight at 4°C)

• Sample preparation considerations:

  • Minimize time between tissue collection and fixation

  • Ensure optimal fixation duration (avoid over or under-fixation)

  • Consider fresh-frozen sections for highly sensitive applications

• Controls:

  • Include tissue known to express ACTN1 as positive control

  • Use ACTN1 knockout or knockdown samples as negative controls

How might ACTN1 antibodies be utilized in studying ACTN1's role in extracellular vesicles and intercellular communication?

Emerging applications for ACTN1 antibodies in extracellular vesicle research:

• EV cargo analysis:

  • Use ACTN1 antibodies to immunoprecipitate and characterize ACTN1-containing EVs

  • Employ western blotting to quantify ACTN1 in isolated EV fractions

• Functional transfer studies:

  • Track ACTN1 transfer between cells via EVs using labeled antibodies

  • Investigate cytoskeletal changes in recipient cells following uptake of ACTN1+ EVs

• Biomarker development:

  • Assess ACTN1+ EVs as potential circulating cancer biomarkers

  • Correlate EV ACTN1 content with disease progression and prognosis

• Therapeutic implications:

  • Explore targeting ACTN1 in EVs to modify intercellular communication

  • Study how therapeutic interventions affect ACTN1+ EV production

This approach builds on ACTN1's established role in cancer progression and cell-cell interactions , extending to the emerging field of EV-mediated intercellular communication.

What are the cutting-edge approaches for studying ACTN1's role in mechanotransduction?

Advanced techniques for investigating ACTN1 in mechanotransduction:

• Live-cell tension sensing:

  • Apply FRET-based sensors to measure mechanical forces across ACTN1

  • Visualize real-time ACTN1 dynamics during mechanical stimulation

• Spatial resolution techniques:

  • Employ super-resolution microscopy (STORM, PALM, SIM) to visualize nanoscale ACTN1 organization

  • Analyze ACTN1 recruitment to mechanosensitive structures

• Mechanical manipulation methods:

  • Use atomic force microscopy to locally apply forces while monitoring ACTN1

  • Apply substrate stretching to analyze ACTN1 redistribution

• Signaling pathway integration:

  • Investigate how ACTN1 connects mechanical stimuli to YAP/TAZ nuclear translocation

  • Examine ACTN1's interaction with integrin-linked kinases during force transmission

This research direction capitalizes on ACTN1's fundamental role as a cytoskeletal protein that anchors actin to various structures and potentially transduces mechanical signals from the extracellular environment to signaling cascades .