ACTN1 Antibody

What are the most reliable techniques for ACTN1 antibody validation prior to experimental use?

Validation of ACTN1 antibodies should follow a multi-step approach to ensure specificity and reliability:

• Western blotting validation: Use cell lysates known to express ACTN1 (such as HepG2, HeLa, or SVT2 cell lines) and look for a specific band at approximately 103 kDa . Compare with positive and negative controls.

• Orthogonal validation: Correlate antibody detection with RNA-seq data to confirm specificity. This enhanced validation approach helps verify that the antibody detects the intended protein rather than off-targets .

• Immunohistochemical cross-validation: Test antibody performance across multiple tissue types. ACTN1 expression patterns should be consistent with its known distribution in tissues .

• Genetic knockdown validation: Use siRNA or CRISPR to reduce ACTN1 expression and confirm corresponding reduction in antibody signal .

• Cross-reactivity testing: Verify species reactivity claims made by manufacturers, particularly if working with non-human models .

What are the key differences between monoclonal and polyclonal ACTN1 antibodies for specific research applications?

The choice between monoclonal and polyclonal ACTN1 antibodies depends on the experimental goals:

For critical quantitative studies, using both antibody types to cross-validate findings is recommended.

How should immunohistochemistry protocols be optimized for ACTN1 detection in different tissue contexts?

ACTN1 immunohistochemistry requires careful optimization based on tissue type and fixation:

• Tissue preparation: For paraffin-embedded sections, use 5-μm thick sections, deparaffinize with xylene and ethanol, followed by heat-induced epitope retrieval in 0.1 mol/L citrate buffer (pH 6.0) by microwaving for 15 minutes .

• Blocking and antibody dilution: Block with 10% (v/v) BSA to inhibit non-specific binding. For monoclonal antibodies like OTI7A4, use dilutions of 1:100-200 for IHC . For polyclonal antibodies like HPA006035, use 1:500-1:1000 .

• Detection system optimization: Use DAB substrate liquid for chromogenic detection and counterstain with hematoxylin. For fluorescent detection, select appropriate secondary antibodies that minimize cross-reactivity .

• Tissue-specific considerations: For hepatocellular carcinoma tissues, ACTN1 shows predominantly cytoplasmic distribution with increased expression compared to non-cancerous liver tissues .

• Validation standards: Always include positive control tissues (such as skeletal muscle) and negative controls (primary antibody omission) to validate staining specificity.

What methodological approaches are recommended for investigating ACTN1's role in cancer progression using antibody-based techniques?

Research on ACTN1's role in cancer progression requires sophisticated experimental designs:

What are the optimal protocols for co-immunoprecipitation studies involving ACTN1 and its binding partners?

Co-immunoprecipitation (Co-IP) studies of ACTN1 interactions require attention to preserve protein complexes:

• Cell lysis conditions: Use gentle lysis buffers containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, and protease/phosphatase inhibitors to maintain protein interactions.

• Antibody selection: Choose antibodies targeting different domains of ACTN1 depending on the interaction being studied. For MOB1-ACTN1 interactions, antibodies against ACTN1 (Abcam, ab50599) and MOB1 (Cell Signaling Technology, #13730) have been successfully used .

• Binding and washing: Incubate lysates with antibody-conjugated beads overnight at 4°C with gentle rotation. For washing, use buffers with decreasing salt concentrations to preserve specific interactions.

• Verification approaches: Confirm interactions through:

  • Reciprocal Co-IP (pull down with anti-MOB1, blot for ACTN1 and vice versa)

  • Immunofluorescence co-localization studies in cell lines such as MHCC-97H cells

  • Functional validation using inhibitors or dominant-negative constructs

• Controls: Always include IgG isotype controls, input samples (5-10% of total lysate), and when possible, samples with known interaction partners altered through mutation or deletion.

How should immunofluorescence protocols be designed for optimal ACTN1 visualization in different cell types?

Immunofluorescence studies with ACTN1 antibodies require specific protocols for different cell types:

• Fixation optimization: For most cell types, use 4% paraformaldehyde for 15 minutes at room temperature. For skeletal myocytes, 100% methanol fixation for 5 minutes may better preserve certain epitopes .

• Permeabilization: Use 0.05% (v/v) Triton X-100 for 1 minute at room temperature for standard cell lines. For highly structured cells like myocytes, increase permeabilization time to 3-5 minutes .

• Antibody dilution and incubation: For primary antibodies:

  • ab18061: Use at 5 μg/mL for iPSC-derived skeletal myocytes

  • ab50599: Dilute 1:100 for cancer cell lines
    Incubate overnight at 4°C for optimal signal-to-noise ratio .

• Co-staining considerations: When performing co-localization studies:

  • Use spectrally distinct fluorophores (e.g., Alexa Fluor 594 for ACTN1 and Alexa Fluor 488 for binding partners)

  • Include counterstains such as DAPI (nuclear) or phalloidin (F-actin)

• Imaging parameters: Capture signals using confocal microscopy with appropriate exposure settings to avoid signal saturation. Z-stack acquisition is recommended for accurate co-localization analysis .

How can ACTN1 antibodies be utilized to investigate ACTN1-related thrombocytopenia (ACTN1-RT)?

ACTN1-related thrombocytopenia (ACTN1-RT) research requires specialized approaches:

• Patient sample analysis: Collect blood samples from individuals with inherited thrombocytopenia of unknown origin. Use mutational screening of ACTN1 by whole-exome sequencing or Sanger sequencing to identify potential pathogenic variants .

• Platelet morphology assessment: Analyze platelet size and structure using blood smears and immunofluorescence with ACTN1 antibodies to detect abnormalities in cytoskeletal organization.

• Functional studies: Evaluate the effects of ACTN1 mutations on:

  • Platelet production using megakaryocyte cultures

  • Platelet function using aggregation assays

  • Cytoskeletal organization using high-resolution microscopy

• Segregation analysis: Perform family studies using ACTN1 antibodies in combination with genetic testing to evaluate inheritance patterns and phenotypic expression of ACTN1 variants .

• Bioinformatic analysis: Utilize predictive tools to assess the pathogenicity of identified ACTN1 variants and correlate with observed phenotypes in patient samples.

What methods should be employed to investigate ACTN1's role in Hippo signaling suppression within hepatocellular carcinoma?

To study ACTN1's tumor-promoting function through Hippo pathway suppression:

• Expression analysis: Perform immunohistochemistry on HCC tissue microarrays (n = 157) with ACTN1 antibodies to establish expression patterns compared to non-cancerous liver tissues .

• Signaling pathway assessment: Use Western blotting to detect changes in:

  • 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)

• Protein interaction studies: Evaluate ACTN1-MOB1 interactions using:

  • Co-immunoprecipitation with appropriate antibodies

  • Confocal microscopy for co-localization analysis

  • Proximity ligation assays for detecting in situ protein interactions

• Functional validation: Assess the effects of:

  • ACTN1 knockdown on tumor growth in xenograft models

  • Pharmacological inhibition of YAP with compounds like verteporfin or super-TDU

  • Reconstitution experiments with wild-type vs. mutant ACTN1

• Clinical correlation: Analyze the relationship between ACTN1 expression and patient survival data using Kaplan-Meier analysis to establish prognostic significance .

What strategies should be implemented to validate antibody specificity when studying ACTN1 in the presence of other alpha-actinin isoforms?

ACTN1 specificity validation in the presence of other alpha-actinin isoforms requires comprehensive approaches:

• Epitope selection analysis: Review the immunogen sequence used to generate the antibody. For instance, HPA006035 targets a sequence specific to ACTN1: "SAKEGLLLWCQRKTAPYKNVNIQNFHISWKDGLGFCALIHRHRPELIDYGKLRKDDPLTNLNTAFDVAEKYLDIPKMLDAEDIVGTARPDEKAIMTYVSSFYHAFSGAQKAETAANRICKV" .

• Sequence alignment comparison: Perform bioinformatic analysis to identify regions of divergence between ACTN isoforms (ACTN1-4) and ensure antibody targets these unique regions.

• Isoform-specific knockout controls: Use CRISPR/Cas9 to individually knockout ACTN1, ACTN2, ACTN3, and ACTN4 in appropriate cell lines, then test antibody reactivity.

• Recombinant protein testing: Validate antibody specificity using purified recombinant proteins of all four ACTN isoforms in parallel Western blots.

• Tissue panel screening: Test antibody performance in tissues with known differential expression of ACTN isoforms:

  • ACTN1/4: Widely expressed in non-muscle tissues

  • ACTN2/3: Predominantly expressed in muscle tissues

What experimental designs best determine the accuracy and reliability of ACTN1 quantification in clinical samples?

Accurate ACTN1 quantification in clinical samples requires rigorous methodological approaches:

• Standardization of sample processing:

  • For tissue samples: Standardize fixation times (e.g., 24h in 10% neutral buffered formalin) and processing protocols

  • For cell samples: Establish consistent lysis procedures and protein extraction methods

• Quantitative assay development:

  • For Western blotting: Use internal loading controls (β-actin, Abcam, ab8227, 1:1000)

  • For immunohistochemistry: Develop scoring systems (e.g., H-score, Allred score) for consistent evaluation

• Calibration curve implementation:

  • Prepare standard curves using recombinant ACTN1 protein

  • Use quantitative ELISA or Western blot densitometry to establish the relationship between signal intensity and protein quantity

• Inter-laboratory validation:

  • Test the same samples across different laboratories using the same antibody lot

  • Calculate coefficients of variation to assess reproducibility

• Clinical correlation studies:

  • Correlate ACTN1 levels with clinicopathological parameters as previously demonstrated with alpha-fetoprotein levels, tumor thrombus, tumor size and TNM stage in HCC