ACTN1 Antibody

What are the principal applications for ACTN1 antibodies in laboratory research?

ACTN1 antibodies serve numerous critical applications in biomedical research. Western blotting represents a fundamental application, allowing researchers to detect and quantify ACTN1 protein expression levels in cell or tissue lysates. Typical protocols utilize antibody dilutions ranging from 1:1000 (as seen with monoclonal antibodies like AT1D10) to 1:200 for some applications . Immunohistochemistry (IHC) enables visualization of ACTN1 distribution in paraffin-embedded tissue sections, revealing its spatial organization within tissues. This application has been particularly valuable in analyzing ACTN1 expression in clinical HCC samples, requiring careful optimization of antigen retrieval methods using citrate buffer (pH 6.0) . Immunofluorescence and immunocytochemistry permit subcellular localization studies of ACTN1, often employing dilutions around 1:100 for optimal staining . Additionally, flow cytometry applications allow researchers to quantify ACTN1 expression at the single-cell level, requiring approximately 2-5 μg antibody per 10^6 cells for effective detection . Each application demands specific optimization to ensure reliable results while minimizing background signal.

How can researchers distinguish between different ACTN1 antibody types and select the appropriate one?

Researchers must consider several critical factors when selecting between monoclonal and polyclonal ACTN1 antibodies. Monoclonal antibodies like clone 1E12 (mouse IgM isotype) and AT1D10 (mouse IgG2a kappa isotype) recognize specific epitopes, offering high specificity but potentially limited sensitivity . These antibodies typically recognize conserved regions, demonstrating cross-reactivity across multiple species including human, mouse, chicken, canine, and quail samples . In contrast, polyclonal antibodies like HPA006035 (rabbit-derived) recognize multiple epitopes, offering enhanced sensitivity for low-abundance targets but with potential increases in background signal . The immunogen used for antibody production represents another crucial selection criterion. For instance, AT1D10 was developed against recombinant human ACTN1 (amino acids 1-249) purified from E. coli, making it especially suitable for N-terminal domain investigations . Meanwhile, 1E12 was generated using a crude homogenate of embryonic dorsal aorta, potentially offering recognition of native protein conformations . Application requirements should ultimately guide selection—monoclonals like AT1D10 have validated compatibility with ELISA, Western blot, immunocytochemistry, and flow cytometry, while others like 1E12 are recommended specifically for immunofluorescence, immunohistochemistry, and Western blot applications .

What methodology should researchers employ to validate ACTN1 antibody specificity?

Rigorous validation of ACTN1 antibody specificity requires a multi-faceted approach combining complementary techniques. Western blot analysis represents a fundamental validation method, where researchers should observe a single distinct band at approximately 103 kDa when probing lysates from cells known to express ACTN1, such as PC-3 or A549 cell lines . Multiple protein loading concentrations (typically 20-40 μg) should be tested to establish detection sensitivity thresholds . Immunoprecipitation followed by mass spectrometry provides definitive validation by confirming the identity of the antibody's target protein through peptide sequence analysis . Knockdown or knockout validation experiments serve as critical controls—comparing staining patterns in wild-type versus ACTN1-silenced cells can definitively demonstrate specificity . For cross-reactivity testing, antibodies should be evaluated against samples from multiple species if the epitope is sufficiently conserved, as demonstrated with antibody 1E12, which shows reactivity across canine, chicken, human, monkey, mouse, quail and shark tissues . Researchers should also perform epitope mapping through peptide array analysis or deletion mutant testing to precisely identify the antibody's recognition site. Enhanced validation approaches, such as orthogonal RNAseq correlation (as employed for HPA006035), provide additional confidence by demonstrating concordance between protein detection levels and transcript abundance data . Importantly, negative controls employing isotype-matched irrelevant antibodies (such as mouse monoclonal IgG for flow cytometry applications) must be included in all validation experiments .

What protocol optimizations are essential when using ACTN1 antibodies for immunohistochemistry?

Successful immunohistochemical detection of ACTN1 in tissue samples requires meticulous attention to several critical protocol parameters. Antigen retrieval methodology significantly impacts staining quality—heat-induced epitope retrieval using 0.1 mol/L citrate buffer (pH 6.0) with microwave treatment for 15 minutes has been validated for optimal ACTN1 epitope exposure in paraffin-embedded sections . Following retrieval, endogenous peroxidase activity must be quenched through incubation in 3% hydrogen peroxide for approximately 20 minutes to prevent non-specific signal development . Blocking protocols utilizing 10% bovine serum albumin (BSA) have proven effective at minimizing background staining while preserving specific ACTN1 signals . Primary antibody concentrations require careful titration—for formalin-fixed, paraffin-embedded (FFPE) tissues, antibody 1E12 has demonstrated optimal results, while a dilution ratio of 1:200 has been validated for ACTN1 antibody ab50599 with overnight incubation at 4°C . Detection systems employing 3,3'-diaminobenzidine (DAB) substrate provide sufficient sensitivity for visualizing ACTN1 expression patterns in hepatocellular carcinoma tissues, with hematoxylin counterstaining enabling clear cellular context . When analyzing results, researchers should note that ACTN1 typically displays cytoplasmic localization patterns in tumor cells, with overexpression particularly evident in tumor tissues compared to adjacent non-tumorous tissues . Comprehensive scoring systems incorporating both staining intensity and percentage of positive cells provide quantitative assessment of ACTN1 expression levels for statistical correlation with clinicopathological parameters.

What considerations are important when using ACTN1 antibodies for protein-protein interaction studies?

Investigating ACTN1 protein-protein interactions requires careful selection of techniques and methodological considerations. Co-immunoprecipitation (Co-IP) represents a foundational approach for studying ACTN1 interactions, where researchers must consider antibody orientation—using anti-ACTN1 for pulldown versus using antibodies against potential binding partners—to avoid epitope masking . For immunofluorescence co-localization studies examining ACTN1's interaction with proteins like MOB1, careful antibody selection is essential to prevent cross-reactivity between secondary antibodies; for instance, documented protocols have successfully combined rabbit-derived anti-MOB1 antibodies (diluted 1:100) with mouse-derived anti-ACTN1 antibodies (diluted 1:100) detected using species-specific Alexa Fluor 594-conjugated anti-rabbit and Alexa Fluor 488-conjugated anti-mouse secondary antibodies . Proximity ligation assays (PLA) offer enhanced sensitivity for detecting transient or weak interactions by producing fluorescent signals only when proteins are within 40nm proximity. When employing fluorescence resonance energy transfer (FRET) techniques, researchers must consider the large molecular size of ACTN1 (103 kDa) when designing fluorophore fusion constructs to prevent steric hindrance . For pull-down assays with recombinant proteins, bacterial expression systems may not recapitulate post-translational modifications that could influence interactions. Researchers should validate all interaction findings through multiple complementary methods, as each technique has inherent limitations that might affect interpretation of ACTN1's diverse binding partnerships, particularly within complex signaling networks like the Hippo pathway .

How can ACTN1 antibodies elucidate its role in the Hippo signaling pathway in cancer?

Investigating ACTN1's role in Hippo signaling requires sophisticated experimental designs employing multiple antibody-dependent techniques. Immunoprecipitation assays using anti-ACTN1 antibodies followed by immunoblotting for Hippo pathway components (particularly MOB1, LATS1, and YAP) can reveal direct protein-protein interactions and potential regulatory complexes . Such experiments have demonstrated that ACTN1 competitively interacts with MOB1, consequently decreasing phosphorylation of downstream effectors LATS1 and YAP . Proximity ligation assays utilizing paired ACTN1 and MOB1 antibodies can visualize and quantify endogenous protein interactions in situ, providing spatial context for these regulatory mechanisms. Chromatin immunoprecipitation (ChIP) experiments employing both ACTN1 and YAP antibodies can determine whether ACTN1-mediated YAP regulation affects transcriptional activity at specific genomic loci. Phospho-specific antibodies against LATS1 (Thr1079) and YAP (Ser127) are essential for monitoring Hippo pathway activation status in response to ACTN1 manipulation . For functional studies, researchers should combine ACTN1 knockdown approaches with pharmacological YAP inhibition using agents like verteporfin or super-TDU, while monitoring pathway activity using validated antibodies . Immunofluorescence co-localization studies can track subcellular distribution patterns of both ACTN1 and Hippo pathway components under various cellular conditions. These approaches have collectively revealed that ACTN1 functions as a tumor promoter by suppressing Hippo signaling activity through physical interaction with MOB1, ultimately promoting YAP-dependent transcriptional programs that enhance hepatocellular carcinoma progression .

What methodological strategies are effective for studying ACTN1 in tumor microenvironment contexts?

Investigating ACTN1 within complex tumor microenvironments requires integration of multiple technical approaches. Multiplex immunohistochemistry or immunofluorescence represents a powerful methodology for simultaneous visualization of ACTN1 alongside cell type-specific markers (CD45 for immune cells, CD31 for endothelial cells, α-SMA for fibroblasts), enabling spatial relationship analysis between ACTN1-expressing tumor cells and stromal components . Sequential chromogenic IHC on serial sections provides an alternative approach when spectral overlap concerns exist. Single-cell RNA sequencing paired with protein validation using flow cytometry and ACTN1 antibodies can identify specific cell populations expressing ACTN1 within heterogeneous tumor tissues. Laser capture microdissection combined with western blotting using ACTN1 antibodies allows quantitative analysis of regional protein expression differences between tumor nests, invasive fronts, and surrounding stroma. For three-dimensional contexts, organoid models stained with ACTN1 antibodies enable visualization of protein localization in architecturally relevant structures. Co-culture systems combining ACTN1-modified tumor cells with stromal components can reveal paracrine signaling effects when analyzed with phospho-specific antibodies against downstream effectors. These approaches have collectively demonstrated that ACTN1 upregulation in HCC correlates with specific microenvironmental features, including increased alpha-fetoprotein levels and presence of tumor thrombus . Critically, ACTN1 antibody-based detection in tissue microarrays has linked expression patterns to clinicopathological parameters and patient outcomes, revealing associations between elevated ACTN1 levels and adverse prognostic indicators including advanced TNM stage .

What techniques can detect post-translational modifications of ACTN1 using specialized antibodies?

Detecting post-translational modifications (PTMs) of ACTN1 requires specialized antibodies and sophisticated analytical approaches. Phosphorylation-specific antibodies targeting known modification sites can be employed in western blotting to quantify relative phosphorylation levels under different cellular conditions. These antibodies must be extensively validated using phosphatase treatments as negative controls and phospho-mimetic mutants as positive controls. Two-dimensional gel electrophoresis followed by western blotting with pan-ACTN1 antibodies can separate differentially modified ACTN1 isoforms based on charge and molecular weight shifts. Mass spectrometry approaches including immunoprecipitation with ACTN1 antibodies followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) provide comprehensive PTM mapping, capable of identifying phosphorylation, acetylation, ubiquitination, and other modifications simultaneously. Phospho-proteomic enrichment using titanium dioxide (TiO2) or immobilized metal affinity chromatography (IMAC) prior to LC-MS/MS analysis enhances detection sensitivity for low-abundance phosphorylated ACTN1 species. For spatial visualization of modified ACTN1, proximity ligation assays combining pan-ACTN1 antibodies with modification-specific antibodies enable in situ detection with subcellular resolution. Phos-tag SDS-PAGE followed by western blotting with standard ACTN1 antibodies offers enhanced separation of phosphorylated variants without requiring phospho-specific antibodies. These approaches collectively enable researchers to elucidate how various signaling pathways modulate ACTN1 function through post-translational modifications, potentially affecting its interactions with binding partners like MOB1 in the Hippo signaling pathway .

How can researchers address cross-reactivity challenges when studying ACTN isoforms?

Addressing cross-reactivity challenges between the four alpha-actinin isoforms (ACTN1-4) requires rigorous experimental design and validation. Pre-adsorption tests represent a crucial validation step—incubating the ACTN1 antibody with excess purified recombinant ACTN1 protein before immunostaining or blotting should eliminate specific signals while leaving any cross-reactive signals intact. Knockout/knockdown validation provides definitive specificity assessment—testing antibodies on samples where ACTN1 has been specifically depleted through CRISPR-Cas9 deletion or siRNA knockdown while other isoforms remain expressed . Peptide competition assays using synthetic peptides corresponding to unique regions of ACTN1 can confirm epitope specificity. Researchers should analyze antibody performance across tissue panels known to differentially express ACTN isoforms—comparing staining patterns in smooth muscle (ACTN1-rich) versus skeletal muscle (ACTN3-rich) tissues. Epitope mapping through techniques like peptide arrays or truncation mutants identifies the precise recognition sequence, enabling theoretical cross-reactivity prediction based on sequence homology between isoforms. Western blot analysis of recombinant ACTN1-4 proteins provides direct assessment of antibody discrimination capabilities. For applications requiring absolute isoform specificity, researchers should consider developing antibodies against uniquely spliced exons or non-conserved C-terminal regions. When working with commercially available antibodies like 1E12 or AT1D10, researchers must review all available validation data, including confirmed species reactivity patterns and recommended applications . For quantitative applications, standard curves using purified recombinant proteins of each isoform can determine cross-reactivity percentages and establish correction factors for more accurate ACTN1 quantification.

How should researchers integrate ACTN1 antibody data with other molecular analyses?

Effective integration of ACTN1 antibody-generated data with complementary molecular analyses requires systematic methodological approaches. Correlation analysis between protein expression levels detected via immunohistochemistry or western blotting with ACTN1 mRNA quantification through RT-qPCR enables assessment of transcriptional versus post-transcriptional regulation mechanisms . This approach has revealed approximately three-fold increases in both ACTN1 mRNA and protein levels in hepatocellular carcinoma compared to non-cancerous liver tissues, suggesting primarily transcriptional upregulation . Multi-omics integration strategies combining ACTN1 antibody-based proteomics with phospho-proteomics, transcriptomics, and metabolomics data can position ACTN1 within broader cellular pathways and regulatory networks. Pathway analysis tools applied to such integrated datasets have identified ACTN1's involvement in Hippo signaling suppression through physical interaction with MOB1, subsequently decreasing LATS1 and YAP phosphorylation . Network analysis algorithms can identify protein-protein interaction hubs, with ACTN1 potentially functioning as a connection point between cytoskeletal organization and signaling cascades. Researchers should implement causal inference methods, including genetic manipulation followed by antibody-based detection of pathway components, to distinguish correlative from causative relationships—knockdown studies have demonstrated that ACTN1 functionally suppresses Hippo signaling and promotes tumor growth, with effects reversed by pharmacological YAP inhibition . For clinical correlations, machine learning approaches can integrate ACTN1 expression data from tissue microarrays with patient survival information, identifying potential prognostic signatures for therapeutic stratification .