ACT4 Antibody

What is the optimal fixation method for ACTN4 immunostaining?

When performing immunocytochemistry or immunohistochemistry for ACTN4, 4% paraformaldehyde fixation followed by 0.1% Triton X-100 permeabilization yields excellent results for preserving both cytoplasmic and nuclear ACTN4 localization. This method maintains cellular architecture while allowing antibody penetration to various subcellular compartments where ACTN4 may be present . For tissues, formalin-fixed paraffin-embedded (FFPE) sections work well with heat-mediated antigen retrieval using Tris-EDTA buffer (pH 9.0) . This approach has been validated with antibodies like ab108198, showing consistent staining patterns across human, mouse, and rat tissues.

Which ACTN4 antibody applications are most reliable for detecting endogenous protein?

Western blotting consistently provides reliable detection of endogenous ACTN4 at its predicted molecular weight of 105 kDa across multiple species including human, mouse, and rat samples . Immunocytochemistry and immunohistochemistry also yield robust results when using well-validated antibodies such as rabbit monoclonal [EPR2533(2)] or rabbit polyclonal antibodies against ACTN4 . Flow cytometry can detect intracellular ACTN4 in fixed and permeabilized cells, though optimization of antibody concentration (typically 1:50 to 1:100 dilution) is essential for reducing background . For co-localization studies, immunofluorescence paired with confocal microscopy provides excellent resolution of ACTN4's association with actin stress fibers and other subcellular structures.

How can I confirm the specificity of my ACTN4 antibody?

Antibody specificity should be verified through multiple approaches:

• Use knockout or knockdown validation - antibodies like ab108198 have been tested in ACTN4 knockout cell lines, showing complete absence of signal when the target protein is not expressed .

• Employ immunoblotting with recombinant fragments of ACTN4 versus other actinin isoforms - specifically, using GST fusion proteins containing distinctive regions of actinin-4 compared with corresponding regions of actinin-1 .

• Perform peptide competition assays where pre-incubation of the antibody with the immunizing peptide should abolish specific staining.

• Include multiple antibodies recognizing different epitopes of ACTN4 to confirm consistent staining patterns .

• Validate across multiple experimental systems to ensure reliable detection in your specific model system.

What controls should be included when using ACTN4 antibodies?

A comprehensive control strategy for ACTN4 antibody experiments should include:

• Positive controls: Cell lines or tissues known to express ACTN4 (e.g., human skeletal muscle, A431 cells, or breast carcinoma tissue) .

• Negative controls: ACTN4 knockout cell lines (HAP1 ACTN4 KO cells have been validated) , tissues with minimal ACTN4 expression, or primary antibody omission controls.

• Loading controls: For Western blotting, GAPDH (37 kDa) or alpha-tubulin antibodies serve as reliable loading controls that don't overlap with the 105 kDa ACTN4 band .

• Isotype controls: Particularly important for flow cytometry and immunohistochemistry to assess non-specific binding of the primary antibody's isotype .

• Cross-reactivity controls: Testing the antibody against other actinin family members, especially actinin-1, to ensure isoform specificity .

How can I differentiate between nuclear and cytoplasmic ACTN4 localization in cancer research?

ACTN4's subcellular localization has significant implications for cancer prognosis, with cytoplasmic localization correlating with increased cell motility and poorer outcomes . To effectively differentiate nuclear from cytoplasmic localization:

• Use confocal microscopy with Z-stack acquisition to precisely visualize three-dimensional protein distribution.

• Perform nuclear/cytoplasmic fractionation followed by Western blotting to quantitatively assess distribution between compartments.

• Apply phosphatidylinositol 3-kinase inhibitors as experimental controls, as they induce nuclear translocation of ACTN4 from the cytoplasm .

• Co-stain with cytoskeletal markers (actin) and nuclear markers (DAPI) to establish clear boundaries between compartments.

• Quantify the nuclear-to-cytoplasmic ratio using image analysis software like ImageJ with appropriate threshold settings.

Research has demonstrated that cytoplasmic ACTN4 is highly concentrated in regions where cells are sharply extended and at the edges of cell clusters, suggesting its role in cell migration . This information can be valuable for studying cancer invasion mechanisms.

What experimental approaches can resolve contradictory data about ACTN4's role in cancer progression?

Researchers often encounter contradictory data regarding ACTN4's role in cancer, where it may appear to have both pro-metastatic and tumor-suppressive functions. To resolve these contradictions:

• Perform isoform-specific knockdown and rescue experiments using CRISPR/Cas9 or RNAi with re-expression of wild-type or mutant ACTN4.

• Assess ACTN4 post-translational modifications (phosphorylation, ubiquitination) that may alter function using specific antibodies or mass spectrometry.

• Conduct temporal analysis of ACTN4 expression and localization during cancer progression using time-lapse microscopy combined with fluorescently-tagged ACTN4.

• Analyze ACTN4 interactomes in different cancer contexts using immunoprecipitation followed by mass spectrometry.

• Determine cell-type specific effects through conditional knockout models or cell-specific promoters.

Studies have shown that cytoplasmic localization of ACTN4 correlates with infiltrative histological phenotype and poorer prognosis in breast cancer , suggesting context-dependent functions that must be carefully evaluated in each experimental system.

How can I effectively study ACTN4's actin-bundling activity in vitro and in living cells?

ACTN4's primary function involves actin filament cross-linking and bundling. To study this activity:

• In vitro approaches:

  • Actin co-sedimentation assays: Recombinant ACTN4 is mixed with F-actin, then ultracentrifuged to separate bundled actin (pellet) from unbound protein (supernatant).

  • Actin-agarose binding assays: Incubate 35S-labeled ACTN4 recombinant protein with actin-coupled agarose beads .

  • TIRF microscopy with fluorescently labeled actin and ACTN4 to visualize bundling in real-time.

• Cellular approaches:

  • Live-cell imaging with fluorescently tagged ACTN4 and actin to track co-localization dynamics.

  • FRAP (Fluorescence Recovery After Photobleaching) to analyze ACTN4 mobility and binding kinetics to actin structures.

  • Super-resolution microscopy (STORM, PALM) to visualize nanoscale organization of ACTN4-actin complexes.

Research has demonstrated that ACTN4 binds directly to actin filaments and that this interaction can be detected both with purified proteins and in cell lysates . Unlike actinin-1, which localizes primarily at focal adhesions and adherens junctions, actinin-4 colocalizes with actin stress fibers throughout the cytoplasm and is particularly concentrated in cell protrusions .

What are the optimal methods for studying ACTN4 protein-protein interactions beyond actin binding?

ACTN4 engages in numerous protein-protein interactions beyond actin binding, including associations with transcription factors and junction proteins. To effectively study these interactions:

• Proximity-based labeling approaches:

  • BioID or TurboID fusion proteins to identify proteins in close proximity to ACTN4 in living cells

  • APEX2 tagging for electron microscopy visualization of interaction sites

• Quantitative interaction proteomics:

  • SILAC or TMT labeling coupled with immunoprecipitation and mass spectrometry

  • Cross-linking mass spectrometry to identify direct binding interfaces

• Microscopy-based interaction analysis:

  • FRET (Förster Resonance Energy Transfer) or BRET to detect direct protein-protein interactions

  • PLA (Proximity Ligation Assay) to visualize endogenous protein interactions with nanometer resolution

• Domain mapping:

  • Generate truncation or point mutants of ACTN4 to identify critical interaction domains

  • Use peptide arrays to map specific binding motifs

Research has shown that ACTN4 interacts with the CART complex for vesicular trafficking, with MICALL2 in tight junction assembly, and functions as a transcriptional coactivator for nuclear hormone receptors PPARG and RARA . Additionally, ACTN4 association with IGSF8 regulates immune synapse formation and T-cell activation .

What is the optimal workflow for studying ACTN4 in primary tissue samples?

When investigating ACTN4 in primary tissues, a comprehensive workflow includes:

• Tissue processing and preservation:

  • Fresh-frozen sections maintain optimal antigen integrity but have poorer morphology

  • FFPE sections provide excellent morphology but require optimized antigen retrieval (Tris-EDTA buffer, pH 9.0 works well for ACTN4)

• Staining protocol:

  • For immunohistochemistry: Use rabbit monoclonal antibodies (e.g., [EPR2533(2)]) at 1:150 dilution following heat-mediated antigen retrieval

  • For multi-color fluorescence: Combine ACTN4 staining with markers for cell type, subcellular compartments, or activation states

• Analysis approaches:

  • Quantitative image analysis for subcellular localization (nuclear vs. cytoplasmic ratio)

  • Correlation with clinical parameters (tumor grade, patient outcome)

  • Digital pathology platforms for automated scoring across large cohorts

In breast cancer studies, cytoplasmic localization of ACTN4 showed significant correlation with infiltrative phenotype and poorer prognosis , demonstrating the clinical relevance of careful subcellular localization analysis.

How can I optimize ACTN4 detection in challenging experimental conditions?

Some experimental conditions present challenges for reliable ACTN4 detection. Here are strategies to overcome common issues:

• Low expression levels:

  • Employ signal amplification methods like tyramide signal amplification for immunohistochemistry

  • Use highly sensitive detection systems like SuperSignal West Femto for Western blotting

  • Consider antibody concentration enrichment techniques for immunoprecipitation

• High background issues:

  • Optimize blocking conditions (5% BSA or 5% non-fat dry milk in TBST often works well)

  • Titrate antibody concentrations carefully (typically 1:1000 for Western blot, 1:100-1:250 for ICC/IF)

  • Include additional washing steps with increased salt concentration

• Cross-reactivity concerns:

  • Select antibodies specifically validated against other actinin isoforms

  • Use knockout/knockdown controls to confirm specificity

  • Consider peptide competition assays to verify epitope specificity

• Fixation-sensitive epitopes:

  • Test multiple fixation methods (paraformaldehyde, methanol, acetone)

  • Compare different antigen retrieval approaches for FFPE tissues

  • Consider native-state detection methods where applicable

What experimental design best captures the dynamic relationship between ACTN4 and the actin cytoskeleton?

To effectively study the dynamic relationship between ACTN4 and actin:

• Live cell imaging approaches:

  • Dual-color live-cell imaging with fluorescently tagged ACTN4 and actin

  • Optogenetic approaches to locally activate or inhibit ACTN4 function

  • Micropatterned substrates to control cell shape and analyze ACTN4 recruitment

• Perturbation strategies:

  • Actin cytoskeleton disruptors (cytochalasin D, latrunculin) to analyze ACTN4 redistribution

  • Phosphatidylinositol 3-kinase inhibitors which trigger ACTN4 nuclear translocation

  • Mechanical stimulation (stretching, shear stress) to examine mechanosensitive responses

• Quantitative analysis methods:

  • Automated tracking of ACTN4-positive structures

  • Colocalization coefficient calculation (Pearson's, Mander's)

  • Ratiometric imaging to measure ACTN4:actin stoichiometry changes

Research has shown that actinin-4 is particularly concentrated in regions where cells are sharply extended and at the leading edge of migrating cells , suggesting its importance in cytoskeletal reorganization during cell movement.

What are the most reliable approaches for comparing the functional differences between actinin isoforms?

To effectively compare functional differences between actinin isoforms (particularly actinin-1 vs. actinin-4):

• Isoform-specific tools:

  • Generate isoform-specific antibodies validated against recombinant proteins

  • Develop isoform-selective inhibitors or dominant-negative constructs

  • Create isoform-specific knockout/knockin models

• Comparative localization analysis:

  • Dual immunostaining to directly compare subcellular distribution

  • Biochemical fractionation followed by isoform-specific Western blotting

  • Super-resolution microscopy to examine nanoscale organization differences

• Functional assays:

  • Cell migration assays (wound healing, transwell) with isoform-specific depletion

  • Actin dynamics measurements (FRAP of actin structures) in isoform-depleted cells

  • Mechanical property assessments (atomic force microscopy, traction force microscopy)

• Interaction profile comparison:

  • Compare binding partners through isoform-specific immunoprecipitation

  • Analyze differences in post-translational modifications between isoforms

  • Assess isoform-specific responses to stimuli or stressors

Studies have demonstrated critical differences between actinin-1 and actinin-4: actinin-1 localizes primarily to focal adhesion plaques and adherens junctions, while actinin-4 associates with actin stress fibers throughout the cytoplasm and concentrates in cell protrusions . These distinct localization patterns reflect their different functional roles in cells.

How can I resolve common technical issues with ACTN4 antibody experiments?

What factors influence ACTN4 expression and localization that could confound experimental interpretation?

Several factors can influence ACTN4 expression and localization, potentially confounding experimental results:

• Cell density and contact inhibition:

  • ACTN4 localization changes dramatically between sparse and confluent cultures

  • Cells at the edges of colonies show different ACTN4 distribution compared to central cells

• Cell cycle phase:

  • Expression and localization patterns may vary through different stages of the cell cycle

  • Synchronize cells when comparing different conditions

• Growth factor signaling:

  • PI3K activity regulates ACTN4 nuclear-cytoplasmic shuttling

  • Document serum conditions and growth factor treatments carefully

• Mechanical environment:

  • Substrate stiffness affects cytoskeletal organization and ACTN4 distribution

  • Standardize culture substrate properties across experiments

• Cellular stress responses:

  • Oxidative stress, heat shock, and other stressors may alter ACTN4 localization

  • Control environmental conditions during experiments

Researchers should carefully document and control these variables to ensure reproducible results when studying ACTN4 biology.

How can researchers integrate ACTN4 antibody data with other experimental approaches for comprehensive understanding?

To develop a comprehensive understanding of ACTN4 biology, researchers should integrate antibody-based data with complementary approaches:

• Multi-omics integration:

  • Correlate protein expression/localization data with transcriptomics to assess regulation mechanisms

  • Combine with phosphoproteomics to identify activity-regulating modifications

  • Integrate with interactome data to place ACTN4 in functional networks

• Functional validation:

  • Follow antibody-based observations with genetic manipulation (CRISPR, RNAi)

  • Use rescue experiments with wild-type or mutant ACTN4 to confirm specificity

  • Apply pharmacological modulators of pathways implicated by localization studies

• Computational approaches:

  • Protein structure prediction to model interactions observed in antibody studies

  • Systems biology approaches to integrate ACTN4 into signaling networks

  • Machine learning analysis of image data to identify subtle localization patterns

• Translational connections:

  • Correlate experimental findings with clinical datasets (cancer databases, tissue atlases)

  • Develop tissue microarray analyses to validate findings across patient cohorts

  • Connect cellular phenotypes to disease-relevant outcomes

By integrating these diverse approaches, researchers can move beyond descriptive antibody-based observations to mechanistic understanding of ACTN4 function in normal physiology and disease.

How can ACTN4 antibodies be utilized in cancer biomarker research?

ACTN4 shows promise as a cancer biomarker, with research indicating its cytoplasmic localization correlates with more aggressive phenotypes and poorer prognosis in breast cancer . To effectively utilize ACTN4 antibodies in cancer biomarker research:

• Standardized assessment protocols:

  • Develop scoring systems that quantify cytoplasmic versus nuclear ACTN4 localization

  • Create tissue microarrays with appropriate controls for high-throughput analysis

  • Establish reproducible cutoff values for "high" versus "low" expression

• Multiplex biomarker panels:

  • Combine ACTN4 staining with established prognostic markers

  • Develop multiplex immunofluorescence panels to assess ACTN4 alongside markers of invasion, proliferation, and stemness

  • Correlate with genomic biomarkers for integrated assessment

• Longitudinal studies:

  • Analyze ACTN4 expression/localization changes during disease progression

  • Compare primary tumors with matched metastatic lesions

  • Assess changes in response to therapy

• Liquid biopsy applications:

  • Investigate ACTN4 in circulating tumor cells as a potential minimally invasive biomarker

  • Develop sensitive detection methods for ACTN4-positive circulating cells

Research has shown that the cytoplasmic localization of ACTN4 was closely associated with an infiltrative histological phenotype and correlated significantly with poorer prognosis in breast cancer , highlighting its potential value as a prognostic biomarker.

What new methodologies are emerging for studying ACTN4 dynamics in live cells?

Emerging technologies for studying ACTN4 dynamics in living cells include:

• Advanced imaging approaches:

  • Lattice light-sheet microscopy for high-speed, low-phototoxicity volumetric imaging

  • Super-resolution live-cell imaging (SoRa, RESOLFT) for nanoscale visualization

  • Adaptive optics for deep tissue imaging of ACTN4 in complex environments

• Optogenetic and chemogenetic tools:

  • Photoswitchable ACTN4 constructs to control activity with spatial precision

  • Rapidly inducible degradation systems to acutely remove ACTN4

  • Split protein complementation to visualize specific ACTN4 interactions

• Biosensor technology:

  • FRET-based tension sensors integrated into ACTN4 to measure mechanical forces

  • Conformation-sensitive ACTN4 biosensors to detect activation states

  • Activity-dependent labeling approaches to identify actively engaged ACTN4 molecules

• Correlative microscopy:

  • Live-to-fixed imaging workflows combining dynamic data with antibody-based molecular characterization

  • CLEM (Correlative Light and Electron Microscopy) to connect ACTN4 dynamics with ultrastructural features

These emerging approaches will provide unprecedented insights into the spatiotemporal regulation of ACTN4 and its functional interactions within living cells.

How can researchers leverage ACTN4 antibodies to understand its role in non-cancer diseases?

While ACTN4's role in cancer is well-studied, its involvement in other diseases is emerging as an important research area. To investigate these connections:

• Kidney disease research:

  • Use ACTN4 antibodies to study its expression in podocytes and correlation with proteinuric diseases

  • Analyze mutations in ACTN4 associated with focal segmental glomerulosclerosis

  • Examine how ACTN4-actin interactions are altered in kidney pathologies

• Neurological disorders:

  • Investigate ACTN4 expression in neuronal cells and its potential role in cytoskeletal regulation during development and disease

  • Study potential associations with neurodegenerative conditions involving cytoskeletal abnormalities

• Immune system dysfunction:

  • Analyze ACTN4's role in immune synapse formation using antibody-based imaging

  • Investigate how ACTN4-IGSF8 interactions regulate T-cell activation in autoimmune conditions

• Cardiovascular pathologies:

  • Examine ACTN4 expression in vascular cells during atherosclerosis progression

  • Investigate its potential role in cardiac remodeling during heart failure

By extending ACTN4 research beyond cancer, researchers can uncover novel disease mechanisms and potentially identify new therapeutic targets.