MEAF6 Antibody

What is MEAF6 and what are its known functions?

MEAF6 (MYST/Esa1-associated factor 6) is a protein encoded by the MEAF6 gene in humans. It is also known by several alternative names including EAF6, C1orf149, CENP-28, NY-SAR-91, chromatin modification-related protein MEAF6, and Esa1p-associated factor 6 homolog . MEAF6 is structurally reported to be 21.6 kilodaltons in mass . Functionally, MEAF6 is involved in chromatin modification processes and has been implicated in cancer progression mechanisms. Research indicates that certain splice variants of MEAF6, particularly MEAF6-1, play a role in promoting cell proliferation, anchorage-independent cell growth, invasion, and tumor growth in prostate cancer models .

What splice variants of MEAF6 have been identified and what is their significance?

Two primary splice variants of MEAF6 have been documented in the literature: MEAF6-1 and MEAF6-2. The MEAF6-1 variant has been identified as particularly significant in neuroendocrine prostate cancer (NEPC) development . Research has shown that MEAF6-1 mRNA levels in NEPC samples are approximately 150-fold higher than in adenocarcinoma prostate cancer (AdPC), while MEAF6-2 mRNA levels show no statistically significant difference between these cancer types . The MEAF6-1 variant appears to be specifically regulated by the neuronal RNA splicing factor SRRM4, which is recruited to an intron 5 region next to the 3' splice site for MEAF6-1 . Functionally, MEAF6-1 has been shown to stimulate cancer cell proliferation and invasion through its effects on downstream gene networks, particularly the ID1 and ID3 genes .

What are the recommended applications for MEAF6 antibodies in research?

Based on available product information, MEAF6 antibodies are most commonly used for Western Blot (WB) applications, with many suppliers reporting validated use in this technique . Additional applications include ELISA and immunohistochemistry (IHC) . For optimal experimental planning, researchers should consider that different commercial antibodies may have different validated applications. For instance, some anti-MEAF6 antibodies are reported to work specifically in Western blot and ELISA, while others have been validated for IHC-p (paraffin) applications as well . When selecting an antibody for a specific application, researchers should review the supplier's validation data for that particular application to ensure compatibility with their experimental design.

How can researchers validate MEAF6 antibody specificity in their experimental system?

Validating antibody specificity is critical, especially for less-characterized targets like MEAF6. A comprehensive validation approach should include:

• Positive and negative controls: Use cell lines or tissues known to express or not express MEAF6. Based on published literature, neuroendocrine cell lines such as NCI-H660, NCI-H69, and NCI-H82 show high expression of MEAF6-1, while adenocarcinoma prostate cancer cell lines could serve as comparative controls .

• Knockout/knockdown validation: Perform siRNA knockdown of MEAF6 (as mentioned in the literature ) to confirm antibody specificity. The disappearance or reduction of signal after knockdown strongly supports antibody specificity.

• Molecular weight verification: Confirm that the detected protein band in Western blots appears at the expected molecular weight of approximately 21.6 kDa .

• Cross-reactivity assessment: Test the antibody against related proteins or in species where cross-reactivity might occur. Many suppliers indicate reactivity with human MEAF6, while some also report reactivity with mouse, rat, and other orthologs .

• Recombinant protein controls: When available, use purified recombinant MEAF6 protein as a positive control.

What are the known limitations of currently available MEAF6 antibodies?

A significant limitation reported in the literature is that currently available antibodies cannot differentiate between MEAF6 splicing variants (MEAF6-1 and MEAF6-2) . This presents a particular challenge for researchers interested in studying the specific roles of these variants, especially given the emerging importance of MEAF6-1 in neuroendocrine prostate cancer. Additionally, some research indicates that "immunoblotting and immunohistochemistry assays were unable to recognize endogenous MEAF6 proteins" , suggesting potential sensitivity issues with at least some commercially available antibodies. These limitations highlight the need for developing more specific antibodies capable of distinguishing between splice variants and detecting endogenous MEAF6 with greater sensitivity.

How can researchers overcome the inability to distinguish between MEAF6 splice variants using antibodies?

Since current antibodies cannot differentiate between MEAF6 splice variants , researchers might consider the following alternative approaches:

• RNA-based detection methods: Use variant-specific primers for RT-PCR, qPCR, or RNA-seq to distinguish and quantify different MEAF6 transcript variants. This approach has been successfully employed in studies showing differential expression of MEAF6-1 in NEPC versus AdPC .

• Epitope-tagging strategies: Clone and express tagged versions of specific MEAF6 variants (e.g., with FLAG, HA, or GFP tags) to allow detection with anti-tag antibodies in overexpression studies.

• Custom antibody development: Consider developing custom antibodies against unique regions of specific splice variants. While challenging, this approach might help overcome the current limitations.

• RNA-ChIP assays: As demonstrated in the literature, RNA chromatin immunoprecipitation can be used to study interactions between splicing factors (like SRRM4) and MEAF6 pre-mRNA, providing insights into splicing regulation .

• Functional studies: Assess the functional consequences of manipulating total MEAF6 or specific variants through overexpression or knockdown approaches, using phenotypic readouts like those described for MEAF6-1 (proliferation, invasion, etc.) .

How is MEAF6 implicated in neuroendocrine prostate cancer progression?

Research indicates that the MEAF6-1 splice variant plays a significant role in neuroendocrine prostate cancer (NEPC) progression, including treatment-induced NEPC (t-NEPC) that can develop following androgen receptor pathway inhibitor (ARPI) therapy . The mechanism involves:

• Differential expression: MEAF6-1 is highly expressed in both t-NEPC tumor biopsies and neuroendocrine cell lines of prostate and lung cancers, with levels approximately 150-fold higher in NEPC compared to adenocarcinoma prostate cancer (AdPC) .

• Regulation by SRRM4: The neuronal RNA splicing factor SRRM4 stimulates MEAF6-1 splicing, providing a mechanistic link between neuronal phenotype acquisition and MEAF6-1 upregulation .

• Downstream effects: Rather than directly inducing neuroendocrine differentiation, MEAF6-1 promotes cancer progression by stimulating cell proliferation, anchorage-independent cell growth, invasion, and xenograft tumor growth .

• Molecular pathways: Gene microarray analysis has identified that MEAF6-1 actions are partially mediated through the ID1 and ID3 gene networks, which are associated with cell proliferation, cell cycling, and cell migration/invasion functions . These ID proteins can override tumor suppressor activities, potentially creating conditions similar to genetic inactivation of RB1 and TP53 .

What experimental models are suitable for studying MEAF6 function in cancer?

Based on the literature, several experimental models have been successfully used to study MEAF6 function in cancer, particularly in the context of prostate cancer:

• Cell line models:

  • Prostate cancer cell lines: LNCaP and PC-3 cells have been used for MEAF6-1 overexpression and knockdown studies .

  • Neuroendocrine cell lines: NCI-H660 (prostate NEPC), NCI-H69, and NCI-H82 (lung cancer with neuroendocrine differentiation) show high endogenous expression of MEAF6-1 and can serve as positive controls .

• Xenograft models: MEAF6-1 has been shown to promote xenograft tumor growth, suggesting that xenograft models are suitable for studying its in vivo functions .

• Patient-derived xenografts (PDXs): These have been used to validate MEAF6 variant expression patterns in NEPC versus AdPC .

• Clinical samples: Analysis of tumor biopsies from patients with AdPC versus NEPC has provided valuable insights into the clinical relevance of MEAF6 variant expression .

• Transfection models: Transient transfection of MEAF6-1 expression vectors in prostate cancer cell lines has been used to study its effects on cell behavior and gene expression .

How do MEAF6 antibodies perform in chromatin immunoprecipitation (ChIP) experiments?

While the search results do not specifically address the performance of MEAF6 antibodies in ChIP experiments, this is an important consideration for researchers studying MEAF6's role in chromatin modification. Based on the general understanding of antibody requirements for ChIP:

• Antibody quality for ChIP: ChIP requires antibodies with high specificity and affinity for their native (non-denatured) target. Researchers should select antibodies specifically validated for ChIP applications, which may not be available for all commercial MEAF6 antibodies.

• Challenges with MEAF6 ChIP: Given the reported difficulties with detecting endogenous MEAF6 proteins by immunoblotting and immunohistochemistry , ChIP experiments targeting MEAF6 may face similar challenges regarding sensitivity and specificity.

• Alternative approaches: RNA-ChIP has been successfully used to study SRRM4 recruitment to MEAF6 pre-mRNA , suggesting that similar approaches could be adapted to study MEAF6 interactions with chromatin or other proteins.

• Validation strategies: If attempting MEAF6 ChIP, researchers should include appropriate controls (such as IgG negative controls and positive controls targeting known MEAF6-associated proteins) and validate ChIP-enriched regions using multiple approaches.

What are the emerging techniques for studying MEAF6 protein interactions and functions?

While not explicitly covered in the search results, several advanced techniques could be valuable for studying MEAF6 interactions and functions:

• Proximity labeling methods: BioID or APEX2-based proximity labeling could help identify proteins that interact with MEAF6 in living cells, providing insights into its functional complexes.

• CRISPR-Cas9 genome editing: Generation of MEAF6 knockout or knock-in cell lines could facilitate functional studies and provide important negative controls for antibody validation.

• Single-cell analysis: Given MEAF6-1's association with specific cancer subtypes, single-cell RNA-seq or proteomics could reveal heterogeneity in MEAF6 variant expression and its correlation with cell states.

• Proteomics approaches: Mass spectrometry-based identification of MEAF6-interacting proteins or post-translational modifications could provide insights into its regulation and function.

• Active learning approaches: As mentioned in search result , active learning approaches for antibody-antigen binding prediction could potentially be applied to improve MEAF6 antibody development and characterization, particularly for distinguishing between splice variants.

What protocols should be optimized when using MEAF6 antibodies for Western blotting?

Based on general principles for Western blotting of challenging proteins and the specific challenges reported with MEAF6 detection:

• Sample preparation: Consider using specialized lysis buffers designed for nuclear proteins, as MEAF6 is involved in chromatin modification. Include protease inhibitors to prevent degradation during extraction.

• Protein loading: Since MEAF6 may be expressed at low levels endogenously, optimize protein loading (consider 50-100 μg of total protein) to improve detection chances.

• Blocking conditions: Test different blocking agents (BSA vs. non-fat dry milk) as this can significantly impact antibody performance. BSA may be preferable for phospho-specific antibodies.

• Antibody dilution and incubation: Given the reported difficulties in detecting endogenous MEAF6 , consider longer primary antibody incubation times (overnight at 4°C) and optimize antibody concentration through titration experiments.

• Detection method: Use high-sensitivity detection methods such as enhanced chemiluminescence (ECL) or fluorescence-based detection systems to improve signal detection.

• Positive controls: Include lysates from cells known to express high levels of MEAF6, such as neuroendocrine cell lines for MEAF6-1 .

• Validation controls: Include MEAF6 knockdown or knockout samples as negative controls to confirm antibody specificity.

What considerations should be made when selecting MEAF6 antibodies for different experimental applications?

When selecting MEAF6 antibodies for specific applications, researchers should consider:

• Antibody type: Different applications may benefit from different antibody types. Polyclonal antibodies often provide higher sensitivity but potentially lower specificity, while monoclonal antibodies offer higher specificity but may recognize only a single epitope.

• Target region: Consider antibodies targeting different regions of MEAF6. The search results mention antibodies targeting the N-terminal region (ARP75744_P050, ARP33354_P050) and C-terminal region (ARP33353_P050) , which may perform differently in various applications.

• Cross-reactivity: Consider the required species reactivity. Multiple suppliers offer MEAF6 antibodies with reactivity to human MEAF6, while some also report reactivity with mouse, rat, rabbit, bovine, dog, guinea pig, horse, and zebrafish orthologs .

• Validated applications: Select antibodies specifically validated for your application of interest. The search results indicate that available MEAF6 antibodies have been validated for applications including Western blot, ELISA, and IHC-p .

• Literature validation: Review the scientific literature for successful use of specific MEAF6 antibodies in your application of interest, though note the reported limitations in detecting endogenous MEAF6 proteins .

• Recognition of splice variants: Be aware that currently available antibodies cannot differentiate between MEAF6 splicing variants , which may be a critical limitation depending on your research question.