CNOT9 Antibody

What is CNOT9 and what cellular functions is it involved in?

CNOT9 (also known as RQCD1, RCD1, or CAF40) is a core component of the CCR4-NOT complex, which functions as one of the major cellular mRNA deadenylases. This complex is linked to various essential cellular processes including bulk mRNA degradation . CNOT9 serves as a key protein-protein interaction site within the CCR4-NOT complex, with both concave and convex surfaces that provide binding sites for various factors . Recent research has revealed CNOT9's critical role in cell polarization, as demonstrated by knockdown experiments showing that depletion of CNOT9 substantially impairs morphological transformation capabilities . Beyond its role in RNA metabolism, CNOT9 has been identified as a transcriptional cofactor involved in retinoic acid-regulated cell differentiation and development pathways .

What applications are CNOT9 antibodies validated for?

CNOT9 antibodies have been validated for multiple experimental applications as shown in the following table:

The extensive validation across multiple applications makes CNOT9 antibodies versatile tools for diverse experimental approaches in both basic and translational research settings .

What species reactivity do commercially available CNOT9 antibodies show?

Commercial CNOT9 antibodies have been tested and validated for cross-reactivity with human, mouse, and rat samples . This broad species reactivity is advantageous for comparative studies across model organisms. The cross-species reactivity likely stems from the high conservation of CNOT9 protein structure across mammalian species. When selecting CNOT9 antibodies for experiments, researchers should verify the specific reactivity profile of their chosen antibody, as some antibodies may exhibit different affinities across species .

How should I optimize CNOT9 antibody dilution across different experimental applications?

Optimization of CNOT9 antibody dilution is critical for generating reliable, reproducible results across different experimental applications. The recommended dilution ranges vary significantly by application:

For Western blotting, the optimal dilution typically falls between 1:1000-1:4000 . Start with 1:2000 and adjust based on signal-to-noise ratio. When performing immunoprecipitation, use 0.5-4.0 μg of antibody per 1.0-3.0 mg of total protein lysate . For immunohistochemistry, begin with 1:1000 dilution and titrate between 1:500-1:2000 depending on your tissue type and fixation method .

For immunofluorescence applications, start with 1:400 dilution and adjust within the range of 1:200-1:800 . The optimal dilution may vary based on cell type, fixation method, and permeabilization protocol.

It is strongly recommended to perform a dilution series experiment for each new application, cell type, or experimental condition. Sample-dependent variations necessitate empirical determination of optimal concentrations in each testing system .

What controls should be included when studying CNOT9 in knockdown experiments?

When conducting CNOT9 knockdown experiments, comprehensive controls are essential for reliable interpretation of results. Based on the studies examining CNOT9's role in the CCR4-NOT complex, the following controls are recommended:

• Non-targeting siRNA/shRNA controls: Essential for distinguishing between specific CNOT9 depletion effects and general RNAi responses .

• Rescue experiments: Re-expression of siRNA-resistant wild-type CNOT9 confirms phenotype specificity. This is particularly important given CNOT9's role in cell polarization .

• Multiple siRNA sequences: Using distinct siRNA sequences targeting different regions of CNOT9 mRNA helps confirm phenotype consistency.

• Deadenylase activity controls: Because CNOT9 is part of the CCR4-NOT complex, parallel knockdown of deadenylase subunits (CNOT7/CNOT8) serves as an important functional control. Research has shown that CNOT7/CNOT8 knockdown produced different phenotypes than CNOT9 knockdown, highlighting CNOT9's specific functions .

• Verification of knockdown efficiency: Both protein (Western blot) and mRNA (qRT-PCR) level verification is critical, with antibody-based quantification of knockdown efficiency.

CNOT9-null cell lines can provide valuable negative controls for antibody specificity validation, as demonstrated in studies where these cells showed resistance to UNK-mediated morphological transformation .

How can I use CNOT9 antibodies to study protein-protein interactions within the CCR4-NOT complex?

CNOT9 antibodies are valuable tools for investigating protein-protein interactions within the CCR4-NOT complex. To study these interactions effectively:

For co-immunoprecipitation experiments, use 0.5-4.0 μg of CNOT9 antibody per 1.0-3.0 mg of protein lysate . Recent research has identified CNOT9 as the principal binding site for proteins like UNK on the CCR4-NOT complex . When investigating novel interaction partners, consider both direct and indirect binding relationships.

When analyzing interaction interfaces, combine antibody-based detection with mutational analysis. Research has revealed that CNOT9 has specific binding sites, including W-binding pockets on its convex surface and a concave surface that serves as a protein-protein interaction site . For example, studies using AlphaFold predictions identified that UNK's IDR segment interacts with CNOT9 through both these surfaces .

Proximity ligation assays using CNOT9 antibodies can provide spatial information about interaction partners in situ. When interpreting results, consider that absence of CNOT9 can substantially reduce interactions between other components of the CCR4-NOT complex, as observed with UNK protein .

The stoichiometry of binding is another important consideration - research has shown that some proteins bind in a 2:1 ratio to CCR4-NOT subcomplexes containing CNOT9 .

What are the implications of CNOT9 variants for neurodevelopmental disorder research?

CNOT9 has recently been identified as a novel gene associated with neurodevelopmental disorders and epilepsy . When using CNOT9 antibodies in research related to these conditions:

Recent clinical studies have identified several de novo missense variants in CNOT9—p.(Arg46Gly), p.(Pro131Leu), p.(Arg227His), and p.(Arg292Trp)—associated with developmental delay, intellectual disability, seizures, muscular hypotonia, facial dysmorphism, and behavioral abnormalities .

When designing experiments to study these variants, consider that molecular modeling predicts these mutations reduce protein stability or impair recognition of interaction partners . Functional analyses have already confirmed pathogenic effects for p.(Pro131Leu) and p.(Arg227His) .

For immunodetection of variant proteins, standard CNOT9 antibodies may show different binding affinities depending on whether epitopes overlap with mutation sites. When studying patient-derived samples or model systems expressing these variants, consider using multiple antibodies targeting different CNOT9 epitopes to ensure reliable detection.

Co-immunoprecipitation experiments comparing wild-type and variant CNOT9 can reveal differential interaction patterns with binding partners, providing insight into pathogenic mechanisms. Subcellular localization studies using immunofluorescence can determine if these variants affect CNOT9 distribution, potentially contributing to cellular dysfunction.

What are the recommended antigen retrieval methods for CNOT9 immunohistochemistry?

Effective antigen retrieval is critical for successful CNOT9 immunohistochemistry. Based on validated protocols:

The primary recommended method is heat-induced epitope retrieval (HIER) using TE buffer at pH 9.0 . This alkaline condition effectively unmasks CNOT9 epitopes in formalin-fixed, paraffin-embedded tissues. As an alternative approach, citrate buffer at pH 6.0 can also be used for antigen retrieval, though this may yield different staining intensities depending on tissue type and fixation conditions .

For challenging tissues, optimization of retrieval time is recommended, typically ranging from 10-30 minutes at 95-100°C. When working with rat colon tissue or human stomach cancer tissue, where CNOT9 antibody has been specifically validated, these retrieval conditions have demonstrated positive and specific staining patterns .

It is advisable to include positive control tissues (such as human stomach cancer tissue or rat colon tissue) in each staining run to verify antibody performance under your specific retrieval conditions .

How do I troubleshoot non-specific bands in CNOT9 Western blotting?

When encountering non-specific bands in CNOT9 Western blotting, consider the following troubleshooting approaches:

First, verify you are observing the expected molecular weight. CNOT9 has a calculated molecular weight of 34 kDa, but is typically observed between 30-40 kDa in Western blotting . Post-translational modifications may account for this variation.

To reduce non-specific bands, optimize antibody dilution within the recommended range of 1:1000-1:4000 . Increasing blocking stringency (5% BSA or milk) and adding 0.1-0.3% Tween-20 to wash buffers can reduce background.

For validation of specific bands, consider using CNOT9 knockdown or knockout samples as negative controls . Published studies have successfully used siRNA-mediated knockdown and CNOT9-null cells to confirm antibody specificity .

If problems persist, try alternative primary antibodies targeting different epitopes of CNOT9. Some commercially available antibodies use immunogens based on the full M1-R244 region , while others may target different segments.

What is the optimal sample preparation for detecting CNOT9 in different cellular compartments?

CNOT9 detection across cellular compartments requires careful consideration of sample preparation protocols:

For cytoplasmic detection, where CNOT9 primarily functions as part of the CCR4-NOT complex, standard RIPA buffer extraction is generally effective. When studying nuclear interactions of CNOT9, which is also described as a transcriptional cofactor , use nuclear extraction protocols with appropriate buffers (such as high-salt extraction).

For immunofluorescence applications, paraformaldehyde fixation (4%, 15-20 minutes) followed by Triton X-100 permeabilization (0.1-0.3%, 10 minutes) has been validated for detecting CNOT9 in HepG2 and hTERT-RPE1 cells . When studying potential membrane associations, more gentle permeabilization using 0.1% saponin may preserve relevant structures.

For co-localization studies examining CNOT9's interaction with RNA-binding proteins or other components of RNA metabolism pathways, consider using methanol fixation, which can better preserve RNA-protein interactions. The optimization of these protocols is especially important when studying CNOT9's role in specialized RNA processing compartments.

How can CNOT9 antibodies be used to investigate RNA degradation pathways?

CNOT9 antibodies offer valuable approaches for investigating RNA degradation pathways through the CCR4-NOT complex:

Researchers can use CNOT9 antibodies in RNA immunoprecipitation (RIP) assays to identify mRNAs associated with the CCR4-NOT complex. This approach has successfully identified thousands of mRNA targets in previous studies .

For studying the dynamics of CCR4-NOT complex assembly on target mRNAs, sequential immunoprecipitation using antibodies against CNOT9 and other complex components can reveal the temporal order of recruitment. When investigating deadenylation activity, researchers should note that while CNOT9 is part of the CCR4-NOT complex, studies have shown that suppressing the deadenylase activity via CNOT7/CNOT8 knockdown had minimal impact on certain cellular phenotypes compared to CNOT9 knockdown .

Co-immunoprecipitation coupled with RNA sequencing can identify how CNOT9 contributes to target RNA selection. Importantly, CNOT9 appears to function as a critical scaffold for protein-protein interactions within the complex, with its absence substantially reducing interactions between other complex components .

For in vivo studies of CNOT9's role in RNA metabolism, proximity labeling approaches combined with CNOT9 antibody validation can map the protein interaction landscape around active deadenylation complexes.

What are the considerations for using CNOT9 antibodies in neurodevelopmental disorder research?

When applying CNOT9 antibodies to neurodevelopmental disorder research:

Recent studies have identified de novo variants in CNOT9 (p.(Arg46Gly), p.(Pro131Leu), p.(Arg227His), and p.(Arg292Trp)) in individuals with developmental delay, intellectual disability, seizures, and other neurological symptoms . CNOT9 antibodies can be valuable in characterizing how these variants affect protein function, localization, and interactions.

For patient-derived samples, researchers should consider potential epitope alterations in variant proteins. If the antibody's binding site overlaps with mutation sites, detection efficiency may be compromised. Using antibodies targeting different epitopes can mitigate this issue.

In neuronal cell models, immunofluorescence using CNOT9 antibodies can reveal abnormal localization patterns associated with pathogenic variants. When conducting such studies, include wild-type controls and consider co-localization with neuronal markers to contextualize findings.

For functional studies in neuronal models, combine CNOT9 immunodetection with electrophysiological measurements to correlate protein expression/localization with functional outcomes like seizure susceptibility. Molecular modeling suggests these variants may reduce protein stability or impair protein-protein interactions , which can be directly tested using co-immunoprecipitation with CNOT9 antibodies.

What emerging research directions involve CNOT9 antibodies?

CNOT9 antibodies are increasingly being applied to emerging research frontiers. Recent studies have revealed CNOT9's unexpected role in cell polarization and morphological transformation, independent of the deadenylase activity of the CCR4-NOT complex . This suggests CNOT9 may have functions beyond RNA metabolism that require further investigation.

The discovery of CNOT9's link to neurodevelopmental disorders opens new avenues for understanding brain development and function . Antibody-based studies can help elucidate how CNOT9 variants disrupt normal neuronal development and contribute to conditions like epilepsy.

CNOT9's role as a protein-protein interaction hub within the CCR4-NOT complex, particularly its interaction with RNA-binding proteins like UNK, suggests it may be central to post-transcriptional regulation networks . Advanced proteomics approaches utilizing CNOT9 antibodies could map these interaction networks comprehensively.

Structure-function studies examining CNOT9's W-binding pockets and its concave interaction surface provide targets for developing more specific antibodies to distinguish between different CNOT9 conformational states . Such conformational-specific antibodies could reveal dynamic aspects of CNOT9 function in various cellular contexts.