ACTN4 Antibody

What is ACTN4 and why is it important in cellular function?

ACTN4 (Alpha-actinin-4) is a ubiquitous actin-binding protein that cross-links actin filaments into bundles to form filopodia and plays a crucial role in cytoskeletal organization. It has a calculated molecular weight of 105 kDa, though it may be observed at 100-105 kDa or sometimes as 105 and 80 kDa bands in experimental settings. ACTN4 is not only involved in structural cellular functions but also participates in transcriptional regulation through interactions with nuclear receptors and transcription factors. Its significance extends beyond cytoskeletal organization to include roles in cellular signaling, gene expression, and pathological conditions including kidney disease and viral infections .

How do I select the appropriate ACTN4 antibody for my specific application?

Selection of an ACTN4 antibody should be based on your specific experimental needs and target species. Consider the following factors:

• Application compatibility: Verify the antibody has been validated for your specific application (WB, IHC, IF/ICC, IP, CoIP, or ELISA)

• Species reactivity: Confirm reactivity with your target species (human, mouse, rat, etc.)

• Clonality: Polyclonal antibodies like 19096-1-AP offer broad epitope recognition, while monoclonal antibodies like 66628-1-Ig provide higher specificity

• Published validations: Check for antibodies with demonstrated success in publications similar to your research

For example, antibody 19096-1-AP shows reactivity with human, mouse, and rat samples across multiple applications, while 66628-1-Ig has been validated for WB, IP, IF, and IHC applications .

What are the optimal dilution ratios for different ACTN4 antibody applications?

Optimal dilution ratios vary by application and specific antibody. Based on validated data:

It is recommended to titrate these antibodies in each testing system to obtain optimal results as outcomes may be sample-dependent. Always verify with validation data for your specific experimental conditions .

What are the critical factors for successful Western blot detection of ACTN4?

Successful Western blot detection of ACTN4 requires attention to several key factors:

• Sample preparation: Use appropriate lysis buffers that preserve protein integrity and phosphorylation status if studying post-translational modifications

• Loading control selection: ACTN4 has a high molecular weight (100-105 kDa), so ensure separation with appropriate percentage gels (typically 8-10% SDS-PAGE)

• Transfer conditions: Due to its size, extend transfer time or use semi-dry transfer systems

• Blocking optimization: 5% BSA in TBST is often effective for reducing background

• Antibody dilution: Start with manufacturer recommendations (1:5000-1:50000 for 19096-1-AP)

• Positive controls: Include validated positive controls like HeLa cells, Jurkat cells, or HEK-293 cells where ACTN4 expression has been confirmed

• Expected band size: Look for bands at 100-105 kDa, with potential additional band at 80 kDa in some cell types

If experiencing weak signals, consider longer exposure times, increased antibody concentration, or enhanced chemiluminescence detection systems .

How can I optimize immunofluorescence detection of ACTN4 in different cell types?

For optimal immunofluorescence detection of ACTN4:

• Fixation method: 4% paraformaldehyde for 10-15 minutes at room temperature works well for preserving ACTN4 structure

• Permeabilization: 0.1-0.5% Triton X-100 for 5-10 minutes

• Dilution optimization: Begin with 1:200-1:800 dilution of antibody 19096-1-AP

• Cell-specific considerations:

  • For HeLa and MCF-7 cells: These are validated positive controls

  • For primary cells: May require longer incubation times with primary antibody

• Counterstaining: Consider co-staining with phalloidin to visualize actin filaments, as ACTN4 is an actin-binding protein

• Confocal imaging: Z-stack imaging may better capture the 3D distribution of ACTN4, especially in filopodial structures

If experiencing non-specific staining, increase blocking time (1-2 hours) with 3-5% BSA and include 0.1% Tween-20 in wash buffers .

What controls should be included when studying ACTN4 protein interactions through immunoprecipitation?

When designing immunoprecipitation experiments to study ACTN4 interactions:

• Input control: 5-10% of total lysate prior to immunoprecipitation

• Negative controls:

  • IgG control: Use matching isotype IgG (rabbit IgG for 19096-1-AP or mouse IgG1 for 66628-1-Ig)

  • No-antibody control: Beads alone to assess non-specific binding

• Positive controls:

  • Known ACTN4 interacting proteins (e.g., nuclear receptors like RARα in AT-RA treated samples)

• Validation approaches:

  • Reciprocal IP: Immunoprecipitate with antibody against suspected interacting protein, then detect ACTN4

  • Knockdown control: Perform IP in ACTN4-depleted cells to confirm specificity

• Technical considerations:

  • Use 0.5-4.0 μg antibody per 1.0-3.0 mg of total protein lysate

  • Consider crosslinking strategies for transient interactions

  • For nuclear interactions, prepare nuclear extracts separately

For Co-IP experiments studying ACTN4's interaction with transcription factors like RARα, AT-RA treatment enhances the association and should be considered as an experimental condition .

How can ACTN4 antibodies be used to study focal segmental glomerulosclerosis (FSGS)?

ACTN4 antibodies are valuable tools for studying FSGS, a kidney disease associated with ACTN4 mutations:

• Mutation detection strategy:

  • Western blot analysis of kidney biopsies or podocyte cell lines to compare wild-type versus mutant ACTN4 expression levels and patterns

  • Immunofluorescence to visualize altered subcellular localization of mutant ACTN4 (typically more cytoplasmic than wild-type)

• Functional assessment approaches:

  • Co-immunoprecipitation to assess differences in protein-protein interactions between wild-type and mutant ACTN4

  • Chromatin immunoprecipitation to evaluate transcriptional regulatory differences, particularly with nuclear receptors like RARα

• Experimental considerations:

  • Include both wild-type and FSGS-linked ACTN4 mutant constructs in overexpression studies

  • Compare podocyte morphology and ACTN4 localization using confocal microscopy

  • Assess transcriptional activity differences using reporter assays for nuclear receptor-mediated transcription

Research has shown that FSGS-linked ACTN4 mutants mislocalize to the cytoplasm and lose their ability to associate with nuclear receptors, affecting transcriptional regulation. This dual mechanism may contribute to FSGS pathophysiology .

What is the role of ACTN4 in SARS-CoV-2 infection and how can antibodies help investigate this?

Recent research has identified ACTN4 as a novel antiviral target against SARS-CoV-2. ACTN4 antibodies can be employed in the following research approaches:

• Expression analysis:

  • Western blot to quantify ACTN4 downregulation during SARS-CoV-2 infection

  • qRT-PCR to measure ACTN4 mRNA levels in conjunction with protein expression

  • Immunofluorescence to visualize ACTN4 localization changes during infection

• Mechanistic studies:

  • Co-immunoprecipitation to confirm ACTN4 interaction with viral nsp12

  • Competition assays to investigate ACTN4's role as a competitor for SARS-CoV-2 RNA and RNA-dependent RNA polymerase

• Functional validation:

  • ACTN4 knockdown studies show increased viral protein expression and replication

  • ACTN4 overexpression studies demonstrate inhibition of viral replication

  • Both wild-type SARS-CoV-2 and Omicron BA.5 variant replication are affected by ACTN4 modulation

These approaches have revealed that ACTN4 inhibits SARS-CoV-2 replication by targeting nsp12 for binding, making it a potential therapeutic target for COVID-19 treatment .

How can ACTN4 antibodies be used to investigate cancer-related research questions?

ACTN4 has been implicated in cancer progression, and antibodies can be utilized for cancer research in several ways:

• Expression profiling:

  • IHC analysis of cancer tissues (validated in human breast cancer and lung cancer tissues)

  • Western blot comparison between cancer cell lines and normal tissues

  • Tissue microarray analysis to correlate expression with clinical outcomes

• Metastasis investigation:

  • Immunofluorescence to study ACTN4's role in filopodial formation and cell motility

  • Co-localization studies with other cytoskeletal and adhesion proteins

• Signaling pathway analysis:

  • Co-immunoprecipitation to identify cancer-specific interaction partners

  • Phosphorylation-specific analysis to study post-translational modifications in cancer contexts

• Transcriptional regulation:

  • ChIP assays to study ACTN4's role in regulating cancer-related gene expression

  • Nuclear/cytoplasmic fractionation followed by Western blot to assess compartmentalization in cancer cells

ACTN4 antibodies have been validated in multiple cancer cell lines including MCF-7, HeLa, and LNCaP, making them suitable for comparative cancer studies .

How do m6A modifications affect ACTN4 expression, and what methodologies can be used to study this?

Recent research has uncovered that m6A modification levels on ACTN4 mRNA regulate its expression. To investigate this relationship:

• m6A-specific methodologies:

  • m6A-specific RNA immunoprecipitation (MeRIP) to assess m6A modification levels on ACTN4 mRNA

  • Actinomycin D chase experiments to analyze ACTN4 mRNA stability in the context of m6A writers (WTAP) or erasers (ALKBH5)

  • Ribosome profiling to evaluate translation efficiency of m6A-modified ACTN4 mRNA

• Enzyme manipulation approaches:

  • Knockdown or overexpression of m6A writers (WTAP) and erasers (ALKBH5) followed by ACTN4 protein quantification

  • RT-qPCR analysis of ACTN4 mRNA levels in WTAP knockdown cells

• Stability assessment:

  • Measure relative ACTN4 mRNA levels remaining after actinomycin D treatment in cells with manipulated m6A machinery

  • Compare ribosome loading onto ACTN4 transcripts in cells with altered WTAP expression

Research has shown that depletion of ALKBH5 (an m6A eraser) enhances ACTN4 expression without affecting mRNA levels, while exogenous ALKBH5 decreases ACTN4 expression, suggesting m6A modifications regulate ACTN4 at post-transcriptional levels .

What approaches can resolve contradictory ACTN4 antibody results between different experimental systems?

When facing contradictory results with ACTN4 antibodies across different experimental systems:

• Antibody validation strategies:

  • Knockout/knockdown validation: Test antibody specificity in ACTN4 knockout or knockdown systems

  • Multiple antibody approach: Use different ACTN4 antibodies targeting distinct epitopes

  • Recombinant protein controls: Include purified ACTN4 protein as positive control

• Technical troubleshooting:

  • Buffer compatibility analysis: Test different lysis and sample preparation buffers

  • Epitope masking assessment: Consider whether protein interactions or modifications may mask antibody epitopes

  • Cross-reactivity evaluation: Check for potential cross-reactivity with other alpha-actinin family members

• Cell/tissue-specific considerations:

  • Expression level variations: Some cell types express ACTN4 at lower levels requiring higher antibody concentrations

  • Isoform differences: Consider potential tissue-specific isoforms or post-translational modifications

  • Subcellular localization: ACTN4 can localize to both cytoplasm and nucleus, requiring different extraction methods

• Interpretation guidance:

  • Expected molecular weight variations: ACTN4 may appear at 100-105 kDa or sometimes with an additional 80 kDa band

  • Consider sample preparation impact: Different detergents or fixation methods may affect epitope accessibility

Always refer to validated positive controls for your specific application and cell type: HeLa, Jurkat, HEK-293, and MCF-7 cells are well-validated models for ACTN4 expression studies .

How can ACTN4 antibodies be integrated into multi-omics approaches for comprehensive functional studies?

Integrating ACTN4 antibodies into multi-omics research approaches can provide comprehensive insights:

• Proteomics integration:

  • Immunoprecipitation coupled with mass spectrometry (IP-MS) to identify ACTN4 interactome

  • Proximity labeling (BioID or APEX) combined with ACTN4 antibody validation to map spatial interactome

  • Phospho-specific analysis to correlate ACTN4 post-translational modifications with functional outcomes

• Genomics coordination:

  • ChIP-seq to map ACTN4 genomic binding sites when functioning as a transcriptional co-regulator

  • Integration with RNA-seq data to correlate ACTN4 genomic binding with gene expression changes

  • CRISPR screens with ACTN4 antibody-based phenotypic readouts

• Single-cell applications:

  • Imaging mass cytometry using ACTN4 antibodies to analyze spatial distribution in tissue contexts

  • Single-cell Western blot to assess ACTN4 expression heterogeneity

  • Immunofluorescence combined with RNA-FISH to correlate protein expression with mRNA localization

• Disease model applications:

  • Tissue microarrays with ACTN4 antibody staining correlated with patient outcomes

  • Liquid biopsy analysis for ACTN4 as potential biomarker

  • Personalized medicine approaches using ACTN4 expression patterns to predict treatment response

These integrated approaches can provide systems-level understanding of ACTN4 function in normal physiology and disease contexts, moving beyond isolated protein studies to comprehensive biological insights .

How can ACTN4 antibodies facilitate research into novel antiviral therapeutic strategies?

Based on recent findings regarding ACTN4's role in viral infections, particularly SARS-CoV-2:

• Therapeutic target validation:

  • Immunoblotting to quantify ACTN4 expression changes in response to potential therapeutic compounds

  • Immunofluorescence to visualize changes in ACTN4-viral protein co-localization with drug treatment

  • Co-immunoprecipitation to assess disruption of ACTN4-viral protein interactions by therapeutic candidates

• Screening methodologies:

  • High-content imaging screening using ACTN4 antibodies to identify compounds that restore ACTN4 expression during viral infection

  • ELISA-based screening to identify molecules that enhance ACTN4-nsp12 binding

  • Phenotypic screening with ACTN4 expression as readout

• Mechanistic validation:

  • Combine ACTN4 agonist treatment with viral load quantification

  • Assess ACTN4 expression changes in clinical samples from patients with different disease severity

  • Evaluate ACTN4 expression in response to existing antiviral therapies

This research direction is particularly promising as ACTN4 has been shown to target nsp12 for binding and impede viral replication, with overexpression inhibiting viral replication while depletion enhanced it .

What methodological approaches best elucidate ACTN4's dual role in cytoskeletal organization and transcriptional regulation?

To investigate ACTN4's dual functionality:

• Subcellular localization studies:

  • Immunofluorescence with high-resolution microscopy to visualize cytoplasmic versus nuclear ACTN4

  • Cell fractionation followed by Western blot to quantify distribution between compartments

  • Live-cell imaging with tagged ACTN4 validated by antibody staining to track dynamic translocation

• Structure-function analysis:

  • Domain-specific antibodies to determine which regions mediate cytoskeletal versus transcriptional functions

  • Immunoprecipitation of wild-type versus mutant ACTN4 to compare interaction partners

  • ChIP-seq combined with cytoskeletal co-localization studies to correlate dual functions

• Signaling pathway integration:

  • Phospho-specific antibodies to determine how post-translational modifications regulate functional switching

  • Stimulation experiments (e.g., AT-RA treatment) to observe translocation and functional changes

  • Temporal analysis of ACTN4 localization and function following cellular stimulation

FSGS-linked ACTN4 mutants provide a natural model for studying this dual functionality, as they show both cytoplasmic mislocalization and loss of transcriptional regulatory capabilities with nuclear receptors like RARα .

What are the best practices for validating ACTN4 knockdown or knockout models using antibodies?

Thorough validation of ACTN4 genetic models requires:

• Expression confirmation strategies:

  • Western blot analysis with multiple ACTN4 antibodies targeting different epitopes

  • qRT-PCR correlation with protein expression data

  • Immunofluorescence to assess complete elimination versus partial reduction

• Specificity controls:

  • Include wild-type, heterozygous, and homozygous knockout samples when possible

  • Rescue experiments with re-expression of ACTN4 to verify phenotype specificity

  • Analysis of other alpha-actinin family members to check for compensatory mechanisms

• Functional validation:

  • Assess cytoskeletal organization using phalloidin staining in conjunction with ACTN4 antibodies

  • Evaluate nuclear receptor-mediated transcription in knockout models

  • Compare phenotypes with published ACTN4 mutant models (e.g., FSGS mutations)

• Technical considerations:

  • For siRNA knockdown: Titrate antibody dilutions to detect residual expression

  • For CRISPR knockout: Verify complete protein loss across the entire cell population

  • For inducible systems: Establish time course of protein depletion using quantitative Western blot

These validation approaches have been successfully employed in studies demonstrating ACTN4's role in SARS-CoV-2 replication and cellular signaling pathways .