ACT7 Antibody

What is ACTL7A and why are antibodies against it significant in research?

ACTL7A (Actin-Like 7A) is a member of the actin protein family with specific structural and functional properties. Antibodies targeting ACTL7A are essential tools for investigating protein expression, localization, and function across various biological systems. These antibodies enable researchers to detect and study ACTL7A in different experimental contexts, contributing to our understanding of its biological roles .

Which epitopes of ACTL7A are commonly targeted by research antibodies?

Several key epitopes are targeted by commercially available antibodies, with the most common being the N-terminal region (amino acids 41-67). Other important epitope regions include mid-protein segments (AA 231-280) and C-terminal regions (AA 226-435), as well as antibodies recognizing the full-length protein (AA 1-435). This diversity of epitope targeting provides researchers with flexibility in experimental design based on protein domain accessibility and functional relevance .

What are the standard applications for ACTL7A antibodies in research settings?

ACTL7A antibodies are validated for multiple research applications including Western Blotting (WB), Enzyme-Linked Immunosorbent Assay (ELISA), and Immunohistochemistry (IHC). These applications enable protein detection, quantification, and localization studies. The antibodies are available in various conjugated forms (unconjugated, biotin-conjugated, HRP-conjugated, FITC-conjugated) to accommodate different detection systems and experimental designs .

How do computational models enhance our understanding of ACTL7A antibody binding specificity?

Advanced computational modeling can identify distinct binding modes between antibodies and their ACTL7A epitopes. These models, trained on phage display experimental data, can predict binding affinity and specificity by analyzing antibody-antigen interactions at the molecular level. Such computational approaches enable researchers to understand the structural basis of antibody specificity and predict how mutations might affect binding properties, facilitating more targeted antibody selection for specific research applications .

What strategies can address cross-reactivity challenges with ACTL7A antibodies?

Cross-reactivity with other actin family proteins represents a significant challenge when using ACTL7A antibodies. Computational biophysics-informed models can be employed to design antibodies with customized specificity profiles, either highly specific for ACTL7A alone or intentionally cross-reactive with defined related proteins. These models work by optimizing energy functions associated with binding modes, allowing researchers to minimize binding to unwanted targets while maximizing affinity for ACTL7A. Experimental validation through competitive binding assays with potential cross-reactive proteins is essential for confirming computational predictions .

How might post-translational modifications affect ACTL7A antibody recognition?

Post-translational modifications can significantly alter antibody epitope recognition. Similar to how modifications like citrullination create targets for autoantibodies in rheumatoid arthritis, modifications of ACTL7A might affect antibody binding. Researchers should consider whether their antibodies recognize modified forms (phosphorylated, acetylated, etc.) of ACTL7A, as these modifications can create neo-epitopes or mask existing ones. This consideration is particularly important when studying ACTL7A in different cellular contexts where post-translational regulation may vary .

What protocols optimize ACTL7A antibody performance in Western blotting?

For optimal Western blotting results with ACTL7A antibodies, the recommended protocol includes:

• Sample preparation with complete protease inhibitors to prevent degradation

• Protein separation on 10-12% SDS-PAGE gels

• Transfer to PVDF membranes (preferred over nitrocellulose for this protein)

• Blocking with 5% non-fat milk in TBST for 1 hour at room temperature

• Primary antibody incubation at 1:1000 dilution overnight at 4°C

• Multiple TBST washes (at least 3 × 10 minutes)

• Species-appropriate HRP-conjugated secondary antibody at 1:5000 dilution

• Detection using enhanced chemiluminescence systems

This protocol has been optimized for the N-terminal targeting antibody (AA 41-67) and may require modification for antibodies targeting other epitopes .

How can AI-assisted approaches improve ACTL7A antibody design?

AI-assisted approaches can revolutionize custom ACTL7A antibody development through:

• Protein language models (like ESM) to predict how sequence variations affect binding properties

• Protein folding models (such as AlphaFold-Multimer) to simulate antibody-antigen interactions

• Computational biology software (like Rosetta) to optimize binding interfaces

These approaches enable rational design of antibodies with improved specificity, affinity, and reduced cross-reactivity. The AI-human collaborative "Virtual Lab" concept demonstrates how these technologies can be integrated to design novel antibodies with customized binding profiles, which could be applied to developing ACTL7A antibodies with superior performance characteristics .

What experimental validations are essential for confirming ACTL7A antibody specificity?

A comprehensive validation strategy should include:

• Western blotting comparing ACTL7A-expressing vs. non-expressing tissues/cells

• Immunoprecipitation followed by mass spectrometry to identify captured proteins

• RNA interference or CRISPR knockout controls to confirm signal reduction/elimination

• Peptide competition assays using the immunizing peptide

• Parallel testing with multiple antibodies targeting different ACTL7A epitopes

• Cross-validation with orthogonal techniques (e.g., mRNA expression analysis)

This multi-modal validation approach ensures that experimental observations genuinely reflect ACTL7A biology rather than antibody artifacts .

How should comparative studies of different ACTL7A antibodies be structured?

A systematic approach to comparing ACTL7A antibodies should include:

This structured approach enables objective comparison of antibodies targeting different epitopes (N-terminal vs. C-terminal) or with different conjugates under identical experimental conditions, facilitating selection of optimal reagents for specific research questions .

What control systems are critical for rigorous ACTL7A antibody experiments?

A comprehensive control system for ACTL7A antibody experiments must include:

• Positive controls: Cell lines or tissues with verified ACTL7A expression

• Negative controls:

  • Tissues known to lack ACTL7A expression

  • CRISPR knockout or siRNA knockdown samples

  • Secondary antibody-only controls

• Specificity controls:

  • Peptide competition/blocking experiments

  • Isotype-matched irrelevant antibodies

• Technical controls:

  • Loading controls (β-actin, GAPDH) for Western blotting

  • Tissue processing controls for IHC/IF

This multi-layered control system ensures experimental rigor and enables confident interpretation of results by distinguishing specific signals from technical artifacts .

How can multiplexed approaches be implemented with ACTL7A antibodies?

Effective multiplexed detection strategies with ACTL7A antibodies require:

• Selection of compatible antibody hosts and isotypes to avoid cross-reactivity

• Strategic use of different conjugates (FITC, biotin, HRP) for orthogonal detection

• Sequential immunostaining protocols with complete stripping between rounds

• Spectral unmixing for fluorescent applications to resolve overlapping signals

• Validation of antibody performance in multiplexed vs. single-target detection

These approaches enable simultaneous detection of ACTL7A alongside interacting proteins, subcellular markers, or other proteins of interest, providing contextual information about ACTL7A localization and function within complex biological systems .

What quantitative analysis approaches are appropriate for ACTL7A antibody experimental data?

Quantitative analysis of ACTL7A antibody data requires:

• For Western blotting:

  • Densitometry with normalization to loading controls

  • Linear dynamic range validation

  • Statistical comparison across biological replicates

• For ELISA:

  • Standard curve generation using recombinant ACTL7A

  • Four-parameter logistic regression analysis

  • Determination of EC50 values for comparative studies

• For immunostaining:

  • Quantitative image analysis with defined threshold parameters

  • Cell-by-cell analysis for heterogeneous populations

  • Colocalization coefficients for multiplexed studies

These approaches transform qualitative observations into robust quantitative data suitable for statistical analysis and comparative studies .

How can researchers resolve contradictory findings when using different ACTL7A antibodies?

When different ACTL7A antibodies yield contradictory results, resolution strategies should include:

• Comprehensive antibody validation using the methods described in section 3.3

• Epitope mapping to understand which protein domains each antibody recognizes

• Investigation of potential post-translational modifications affecting epitope accessibility

• Correlation with mRNA expression data using RT-qPCR or RNA-seq

• Mass spectrometry analysis to identify the exact protein species being detected

• Consideration of protein conformation differences between applications

• Functional studies (e.g., immunodepletion) to confirm biological relevance

This systematic troubleshooting approach can reconcile apparently contradictory findings by identifying the biological or technical factors responsible for the discrepancies .

What approaches distinguish between specific and non-specific binding of ACTL7A antibodies?

Distinguishing specific from non-specific binding requires:

• Pre-adsorption studies with immunizing peptide to block specific binding

• Competitive binding assays with excess unlabeled antibody

• Correlation between signal intensity and known ACTL7A expression patterns

• Comparison of staining/detection patterns across multiple antibodies targeting different ACTL7A epitopes

• Signal reduction/elimination in genetic knockdown/knockout systems

• Biochemical validation through techniques like immunoprecipitation followed by Western blotting or mass spectrometry

These complementary approaches provide strong evidence for binding specificity when consistent results are observed across multiple methods .

Data Table: ACTL7A Antibody Characteristics and Applications