ACTL6A Antibody

What is ACTL6A and why is it significant in research?

ACTL6A (actin-like 6A) is a member of the actin-related protein (ARP) family that shares significant sequence identity with conventional actins. The protein is encoded by the ACTL6A gene in humans and has several alternative names including BAF53, ACTL6, ARPN-BETA, Arp4, BAF53A, and 53 kDa BRG1-associated factor A . With a calculated molecular weight of 47.5 kDa (though observed at 45-51 kDa in Western blots), ACTL6A functions as a critical component of the SWI/SNF chromatin remodeling complex .

The significance of ACTL6A in research stems from its essential roles in multiple cellular processes:

• Chromatin remodeling and transcriptional regulation

• Embryonic development and pluripotency maintenance

• Interaction with c-Myc in oncogenic pathways

• Association with epithelial-mesenchymal transition (EMT) in cancer

• Participation in the embryonic SWI/SNF complex (esBAF)

Recent studies highlight ACTL6A's importance in cancer biology, where it appears to function as an oncogene in several malignancies, making it a valuable target for both basic research and potential therapeutic development .

What validated applications exist for ACTL6A antibodies?

ACTL6A antibodies have been validated for multiple experimental applications, with varying degrees of optimization:

When selecting antibodies, researchers should consider the specific application requirements and whether validation data exists for their experimental system. For critical experiments, validating antibody performance in the specific research context is strongly recommended, particularly for applications with limited published validation .

How should researchers distinguish between ACTL6A and ACTL6B in experiments?

ACTL6A and ACTL6B are paralogs with high sequence similarity but distinct biological functions, making specific detection critical:

ACTL6A is widely expressed and primarily associated with the embryonic SWI/SNF complex (esBAF), playing roles in pluripotency and proliferation. In contrast, ACTL6B (BAF53B) is predominantly expressed in neural tissues and appears in the neural-specific SWI/SNF complex (nBAF), contributing to memory formation and synaptic plasticity .

To distinguish between these paralogs, researchers should:

• Use paralog-specific antibodies targeting divergent regions

• Validate antibody specificity with recombinant proteins or knockout controls

• Consider the biological context - ACTL6A associates with stemness while ACTL6B relates to differentiation

• When analyzing both proteins, perform parallel detection with paralog-specific antibodies

• For mRNA analysis, design primers in non-conserved regions

In tissues where both paralogs are expressed (such as developing neural tissues), extra validation steps are essential to prevent cross-reactivity or misinterpretation of results .

What are the recommended dilutions and conditions for ACTL6A antibodies in different applications?

Optimal dilutions vary by application and specific antibody formulation. The following guidelines are based on commonly used commercial ACTL6A antibodies:

For Western blotting, ACTL6A typically appears between 45-51 kDa, though the calculated molecular weight is 47 kDa . For IHC applications, antigen retrieval is critical, with TE buffer (pH 9.0) being the primary recommended method, though citrate buffer (pH 6.0) can serve as an alternative .

These ranges should be considered starting points. Each new experimental system requires optimization, especially when working with different tissue types, cell lines, or fixation methods .

What are the key considerations for immunohistochemistry with ACTL6A antibodies?

Successful immunohistochemistry (IHC) with ACTL6A antibodies requires attention to several critical factors:

• Antigen retrieval: This step is essential for ACTL6A detection. Use TE buffer (10 mM Tris, 1 mM EDTA) at pH 9.0 as the primary method, heating slides to 95-100°C for 15-20 minutes. Citrate buffer (pH 6.0) can serve as an alternative .

• Section preparation: Use 4-5 μm sections for optimal balance between signal strength and resolution.

• Validated tissues: Human lung cancer and skin cancer tissues have been validated as positive controls for ACTL6A IHC .

• Antibody dilution: Start with 1:100 dilution and optimize based on signal-to-noise ratio. The recommended range is 1:50-1:500 .

• Incubation conditions: Overnight incubation at 4°C generally provides optimal specific binding.

• Localization interpretation: ACTL6A primarily shows nuclear localization, consistent with its role in chromatin remodeling complexes. Cytoplasmic staining may occur in certain cancer types but should be critically evaluated .

When establishing IHC protocols for ACTL6A, researchers should include positive control tissues processed simultaneously to confirm staining patterns and optimize protocol parameters.

How can researchers optimize Western blot detection of ACTL6A?

For optimal Western blot detection of ACTL6A, consider the following recommendations:

• Sample preparation: Nuclear extraction may improve detection, given ACTL6A's primary nuclear localization.

• Protein loading: 20-30 μg of total protein per lane is typically sufficient.

• Antibody dilution: Commercial ACTL6A antibodies generally work well at 1:2,000-1:16,000 dilutions .

• Expected band size: ACTL6A appears between 45-51 kDa .

• Positive controls: HeLa, K-562, and MCF-7 cells have been validated as reliable positive controls .

• Blocking conditions: 5% non-fat milk in TBST for 1-2 hours at room temperature typically minimizes background.

• Signal development: Both chemiluminescence and fluorescence-based detection systems work well.

If multiple bands appear, researchers should consider: (a) increasing antibody dilution, (b) using longer blocking steps, (c) increasing washing stringency, or (d) validating with ACTL6A knockdown controls to identify the specific band .

What considerations are important when using ACTL6A antibodies for ChIP experiments?

Chromatin immunoprecipitation (ChIP) with ACTL6A antibodies requires specific optimization:

• Antibody selection: Use only ChIP-validated antibodies; not all WB-validated antibodies work effectively in ChIP applications.

• Crosslinking conditions: Standard 1% formaldehyde for 10 minutes is typically sufficient for ACTL6A ChIP.

• Sonication optimization: Aim for 200-500 bp DNA fragments for optimal resolution.

• Antibody amount: Use 2-5 μg per ChIP reaction; titrate for optimal signal-to-noise ratio.

• Controls: Include IgG negative control and, if possible, ACTL6A knockdown cells as specificity controls.

• Analysis approach: ChIP-seq is preferred over ChIP-qPCR to reveal genome-wide binding patterns.

• Data interpretation: Remember that ACTL6A binding patterns reflect its incorporation into SWI/SNF complexes rather than direct DNA binding.

For the most informative results, consider performing parallel ChIP experiments with other SWI/SNF components (such as BRG1/SMARCA4 or SNF5/SMARCB1) to identify shared binding sites and complex-specific functions .

How should researchers approach studying ACTL6A's role in SWI/SNF complexes?

ACTL6A functions primarily within the context of SWI/SNF chromatin remodeling complexes, requiring specific experimental approaches:

• Complex composition analysis: Immunoprecipitate ACTL6A followed by mass spectrometry or Western blotting for other SWI/SNF components to identify complex-specific associations.

• Functional separation: ACTL6A participates in canonical BAF complexes and embryonic esBAF complexes, but not in PBAF or neuron-specific nBAF complexes (which contain ACTL6B instead) .

• Genomic localization: Compare ChIP-seq profiles of ACTL6A with other SWI/SNF components to identify shared and unique binding sites.

• Developmental dynamics: Track changes in complex composition during differentiation, particularly the ACTL6A-to-ACTL6B switch in neural development.

• Functional redundancy: Consider the potential compensatory roles of ACTL6B in experimental designs, particularly in neural contexts.

When investigating SWI/SNF-related functions, researchers should consider whether their experimental question addresses ACTL6A's individual role or its contribution to complex activity. For mechanistic studies of chromatin remodeling, nucleosome positioning assays or ATAC-seq following ACTL6A manipulation can provide functional insights beyond protein interactions .

What approaches are recommended for studying ACTL6A in cancer research?

ACTL6A has emerged as an important factor in cancer biology, with specific methodological considerations:

• Expression analysis: Use IHC with standardized scoring systems to evaluate ACTL6A expression across cancer types and stages. Digital pathology approaches can improve quantification reliability.

• EMT connection: Investigate ACTL6A's relationship with epithelial-mesenchymal transition by co-staining with EMT markers (E-cadherin, Vimentin, Snail) in cancer progression models.

• Functional validation: Employ knockdown/knockout approaches followed by phenotypic assays (proliferation, migration, invasion) to establish causal relationships.

• Mechanistic investigation: Combine ChIP-seq with RNA-seq after ACTL6A manipulation to identify direct transcriptional targets relevant to cancer phenotypes.

• c-Myc interaction: Investigate the functional collaboration between ACTL6A and c-Myc through co-immunoprecipitation, proximity ligation assays, and co-localization studies .

• Therapeutic implications: Evaluate how ACTL6A expression or complex assembly changes in response to treatment with epigenetic modulators or targeted therapies.

Cancer researchers should be aware that ACTL6A expression patterns can vary significantly between cancer types and stages. Multi-parameter analyses examining ACTL6A in conjunction with other SWI/SNF components and cancer-specific markers will provide more meaningful insights than ACTL6A detection alone .

How can ACTL6A antibodies contribute to pluripotency and developmental research?

ACTL6A's involvement in pluripotency maintenance presents specific considerations for stem cell research:

• Pluripotency network: Analyze ACTL6A in conjunction with core pluripotency factors (OCT4, SOX2, NANOG) through co-immunoprecipitation, co-localization, and ChIP-seq approaches.

• esBAF complex characterization: Use ACTL6A antibodies to immunoprecipitate and analyze embryonic stem cell-specific BAF complexes, which differ from neural progenitor BAF complexes.

• Differentiation dynamics: Track ACTL6A expression and localization during differentiation protocols using Western blotting, immunofluorescence, and ChIP-seq at multiple timepoints.

• ACTL6A-to-ACTL6B switch: Monitor the developmental transition from ACTL6A to ACTL6B incorporation in SWI/SNF complexes, particularly during neural development.

• Reprogramming studies: Investigate ACTL6A's role during induced pluripotent stem cell generation through gain- and loss-of-function approaches.

Developmental biologists should note that ACTL6A functions within the embryonic SWI/SNF (esBAF) complex, which has a distinct composition from neural-specific nBAF complexes (containing ACTL6B). This distinction is critical for understanding lineage-specific chromatin remodeling activities during development .

How can researchers troubleshoot non-specific binding with ACTL6A antibodies?

Non-specific binding is a common challenge when working with ACTL6A antibodies. Several approaches can minimize this issue:

• Multiple bands in Western blot:

  • Increase antibody dilution (try 1:5,000-1:16,000)

  • Use freshly prepared samples

  • Include ACTL6A knockdown controls to identify specific bands

  • Try alternative antibody clones targeting different epitopes

• High background in IHC/IF:

  • Extend blocking time (1-2 hours) with 5-10% normal serum from secondary antibody host

  • Increase antibody dilution (e.g., try 1:200-1:500 if starting with 1:100)

  • Optimize washing steps (increase number and duration)

  • Use alternative antigen retrieval methods

• Non-nuclear staining:

  • Optimize fixation protocol (overfixation can create artifacts)

  • Validate with fractionation experiments

  • Compare with alternative antibodies

• Signal in knockout controls:

  • Try antibodies raised against different epitopes

  • Increase washing stringency

  • Consider potential cross-reactivity with ACTL6B due to sequence similarity

When encountering non-specific binding, researchers should first consult the antibody documentation for known cross-reactivity. Validation with knockout or knockdown controls remains the gold standard for confirming specificity, particularly when working with novel experimental systems .

How should researchers interpret contradictory data from different ACTL6A antibodies?

When different antibodies yield contradictory results, systematic investigation is required:

• Different subcellular localization:

  • Compare with tagged ACTL6A expression (e.g., GFP-ACTL6A)

  • Perform subcellular fractionation followed by Western blotting

  • Validate with siRNA knockdown to determine which signal decreases

• Different molecular weight bands:

  • Perform knockdown experiments to identify which bands represent ACTL6A

  • Consider potential post-translational modifications or isoforms

  • Compare with recombinant ACTL6A protein standard

• Different interaction partners:

  • Validate interactions with alternative methods (proximity ligation, mass spectrometry)

  • Determine if different antibodies might be disrupting specific interactions

  • Consider epitope availability within protein complexes

• Different ChIP binding profiles:

  • Perform sequential ChIP to identify sites bound by multiple antibodies

  • Compare with binding profiles of other SWI/SNF components

  • Validate key binding sites with alternative techniques

The most rigorous approach combines multiple antibodies with orthogonal techniques (such as mass spectrometry, CRISPR/Cas9 editing, or recombinant protein studies) to establish ground truth. For contradictory findings, researchers should consider whether different antibodies might be detecting distinct isoforms, post-translational modifications, or conformation states of ACTL6A .

What quality control measures should be implemented when using new lots of ACTL6A antibodies?

Lot-to-lot variation is a significant challenge in antibody-based research that requires systematic quality control:

• Initial validation:

  • Compare directly with previously validated lot using identical conditions

  • Run side-by-side Western blots with positive control samples (HeLa, K-562, MCF-7 cells)

  • Perform titration experiments to determine optimal working dilution

• Specificity testing:

  • Include ACTL6A knockdown or knockout samples when available

  • Check for cross-reactivity with related proteins, particularly ACTL6B

  • Verify expected molecular weight (45-51 kDa)

• Functional validation:

  • For critical applications, confirm expected subcellular localization

  • For ChIP applications, verify enrichment at known target sites

  • For co-IP experiments, confirm detection of established interaction partners

• Documentation practices:

  • Record lot numbers in laboratory notebooks

  • Include lot information in publications

  • Maintain reference aliquots of well-performing lots for future comparisons

For highly quantitative applications or longitudinal studies, researchers should secure sufficient antibody from a single lot to complete the entire project. When this isn't possible, careful normalization and calibration between lots is essential to maintain data comparability .

How are ACTL6A antibodies being used to investigate epithelial-mesenchymal transition in cancer?

ACTL6A has emerged as a regulator of epithelial-mesenchymal transition (EMT), with antibody-based approaches advancing this research area:

• Expression correlation studies:

  • IHC analysis of ACTL6A in cancer progression models

  • Correlation with EMT markers (E-cadherin loss, Vimentin upregulation)

  • Association with invasion depth and metastatic potential

• Mechanistic investigations:

  • ChIP-seq to identify ACTL6A binding at EMT-related genes

  • Co-IP to detect interactions with EMT transcription factors

  • Validation of direct regulation through reporter assays

• Therapeutic implications:

  • Monitoring ACTL6A levels after treatment with EMT-targeting compounds

  • Assessment of ACTL6A as a biomarker for treatment response

  • Correlation with resistance to conventional therapies

• In vitro models:

  • Immunofluorescence tracking of ACTL6A during EMT induction

  • Analysis of SWI/SNF complex remodeling during transition states

  • Real-time visualization in cell migration/invasion assays

Researchers investigating ACTL6A in EMT should consider combined detection of ACTL6A with established EMT markers to establish correlative or causal relationships. Time-course experiments during EMT induction provide particularly valuable insights into the regulatory relationships and potential intervention points .

What are the latest applications of ACTL6A antibodies in single-cell analysis technologies?

Emerging single-cell technologies offer new opportunities for ACTL6A research:

• Single-cell proteomics:

  • Mass cytometry (CyTOF) using metal-conjugated ACTL6A antibodies

  • Integration with other chromatin regulatory proteins

  • Correlation with cell cycle or differentiation markers

• Chromatin profiling:

  • scCUT&Tag using ACTL6A antibodies for single-cell chromatin binding

  • Identification of cell-specific binding patterns

  • Integration with transcriptomic data

• Spatial biology:

  • Multiplex imaging with cyclic immunofluorescence

  • Co-localization analysis with other SWI/SNF components

  • Spatial relationships in developmental or tumor contexts

• Multi-modal analysis:

  • CITE-seq approaches combining ACTL6A protein detection with transcriptomics

  • Correlation of protein levels with target gene expression

  • Identification of functional cell states

These approaches can reveal heterogeneity within cell populations that bulk analyses would miss. For ACTL6A research, single-cell approaches are particularly valuable for understanding transitional states during differentiation or cancer progression, where ACTL6A levels may vary between individual cells and correlate with cell fate decisions .

How can ACTL6A antibodies contribute to understanding the dynamics of chromatin remodeling complexes?

ACTL6A antibodies enable sophisticated analyses of chromatin remodeling dynamics:

• Complex assembly/disassembly:

  • Quantitative IP-mass spectrometry at different developmental stages

  • Analysis of subunit stoichiometry in different cell types

  • Identification of context-specific interactions

• Genome-wide localization changes:

  • ChIP-seq under different cellular conditions

  • Comparison with nucleosome positioning data

  • Integration with chromatin accessibility maps

• Real-time dynamics:

  • Live-cell imaging with fluorescently tagged SWI/SNF components

  • FRAP (Fluorescence Recovery After Photobleaching) to measure residence time

  • Single-molecule tracking to analyze diffusion and binding kinetics

• Structural insights:

  • Proximity labeling approaches to map protein neighborhoods

  • Cryo-EM studies of immunopurified complexes

  • Analysis of ACTL6A's position within the SWI/SNF architecture

These approaches can reveal how ACTL6A-containing complexes assemble, target specific genomic loci, and modify chromatin structure in different cellular contexts. Combining multiple techniques provides complementary insights into both composition and function of these complex macromolecular machines .