N6AMT2 Antibody

What species reactivity is available for N6AMT2 antibodies?

N6AMT2 antibodies are available with reactivity against multiple species, though the most commonly available are those targeting human, mouse, and rat N6AMT2 proteins . Human-reactive antibodies are the most prevalent, with several commercial options validated specifically for human N6AMT2 detection . Researchers should note that species cross-reactivity can vary significantly between antibody products, even those from the same manufacturer. For example, while some antibodies react with N6AMT2 from all three species (human, mouse, rat), others are species-specific and react only with human N6AMT2 . When selecting an antibody for multi-species studies, it is critical to verify that the epitope region is conserved across the species of interest. Sequence alignment analysis of the antibody's target region can help predict cross-reactivity when experimental validation data is limited.

What applications are N6AMT2 antibodies validated for?

N6AMT2 antibodies have been validated for several common laboratory applications, with the most frequently validated being ELISA (Enzyme-Linked Immunosorbent Assay), WB (Western Blot), and IHC (Immunohistochemistry) . Different antibody products show varying application profiles, with some validated for multiple techniques while others are application-specific. For Western blot applications, polyclonal antibodies raised in rabbits are commonly used, which can detect the approximately 31 kDa N6AMT2 protein . For immunohistochemistry, several antibodies have demonstrated specific staining patterns in tissues including muscle, colon, and pancreatic samples, suggesting their utility for histological studies . Some N6AMT2 antibodies are also available with fluorescent conjugates such as FITC, making them suitable for direct immunofluorescence applications . For advanced applications such as ChIP (Chromatin Immunoprecipitation) or IP (Immunoprecipitation), additional validation is typically required as these applications are more technically demanding.

How should researchers validate the specificity of N6AMT2 antibodies?

Validating N6AMT2 antibody specificity requires a multi-faceted approach to ensure reliable experimental results. First, perform Western blot analysis using both recombinant N6AMT2 protein and cell/tissue lysates, confirming a single band of appropriate molecular weight (approximately 31 kDa) . Second, include proper controls such as lysates from N6AMT2 knockout or knockdown cells, which should show reduced or absent signal compared to wild-type samples . Third, validate antibody specificity using immunoprecipitation followed by mass spectrometry, which can identify all proteins recognized by the antibody . Fourth, conduct peptide competition assays where pre-incubation of the antibody with the immunizing peptide should block specific staining. Fifth, perform immunofluorescence experiments comparing staining patterns in cells with and without N6AMT2 expression, observing for expected subcellular localization. The recent findings of cross-reactivity between N6AMT1 antibodies and Aurora Kinase A highlight the importance of rigorous validation . Even antibodies targeting different methyltransferases can exhibit unexpected cross-reactivity due to shared sequence motifs, emphasizing the need for thorough controls in every experiment using N6AMT2 antibodies.

What potential cross-reactivity issues exist with N6AMT2 antibodies?

While specific cross-reactivity data for N6AMT2 antibodies is limited in the provided search results, important lessons can be drawn from studies of the related methyltransferase N6AMT1. Recent research has demonstrated that several commercial N6AMT1 antibodies cross-react with Aurora Kinase A (AURKA) due to a shared ENNPEE motif present in both proteins . By analogy, N6AMT2 antibodies may exhibit similar "specific non-specificity" with proteins sharing conserved sequence motifs or structural domains characteristic of the methyltransferase family. Potential cross-reactivity candidates include other members of the SAM-binding methyltransferase superfamily, particularly those in the EFM5 family to which N6AMT2 belongs . Researchers should be vigilant for cross-reactivity with other known lysine methyltransferases, especially those involved in EEF1A modification. Additionally, given that N6AMT2 is also known as EEF1AKMT1, antibodies raised against either name might recognize the same protein but could have been validated under different experimental conditions . To mitigate these risks, comprehensive validation using multiple detection methods, knockout/knockdown controls, and careful analysis of unexpected bands or staining patterns is essential.

How can researchers distinguish between N6AMT2 and its aliases in antibody selection?

N6AMT2 is known by several aliases, most notably EEF1AKMT1 (EEF1A lysine methyltransferase 1), which reflects its functional role as a protein N-lysine methyltransferase that catalyzes trimethylation of EEF1A at Lys-79 . Other aliases include 2510005D08Rik, AW045965, and RGD1306762, depending on the species and database . When selecting antibodies, researchers should first cross-reference multiple databases (UniProt, NCBI, etc.) to compile a complete list of aliases for N6AMT2. Next, search for antibodies using all identified aliases, as manufacturers may list products under different nomenclature. Compare the immunogen sequences used to generate each antibody - those raised against identical or overlapping epitopes should recognize the same protein despite different naming conventions. Verify that the antibody's target molecular weight matches N6AMT2 (approximately 31 kDa). For absolute confirmation, test the antibody on recombinant N6AMT2 protein with a known tag, and perform parallel detection with both anti-tag and anti-N6AMT2 antibodies . Finally, consult recent literature to determine which nomenclature is currently preferred in the field, as protein naming conventions evolve with advances in functional characterization.

How can N6AMT2 antibodies be used to study eEF1A methylation patterns?

N6AMT2 antibodies can be strategically employed to investigate eEF1A methylation patterns through multiple approaches. First, researchers can use N6AMT2 antibodies in combination with methyl-specific eEF1A antibodies to correlate N6AMT2 expression levels with eEF1A methylation status at Lys-79 . This can be achieved through dual immunofluorescence staining or sequential Western blots of the same samples. Second, immunoprecipitation with N6AMT2 antibodies followed by mass spectrometry can identify eEF1A as an interaction partner and reveal co-precipitating proteins that may regulate the methyltransferase activity. Third, chromatin immunoprecipitation (ChIP) assays using N6AMT2 antibodies can determine if this methyltransferase associates with actively translating ribosomes. Fourth, N6AMT2 knockdown or knockout experiments followed by Western blot analysis with methyl-specific antibodies can reveal direct causality between N6AMT2 activity and eEF1A methylation patterns . Research has shown that N6AMT2 depletion impacts not only Lys-79 methylation but also affects eEF1AK36me3 levels, suggesting complex crosstalk between different methylation sites . This indicates that N6AMT2 antibodies can help reveal the interdependence of various post-translational modifications on eEF1A, providing insights into the hierarchical regulation of protein translation.

What experimental approaches can determine the impact of N6AMT2 on protein translation?

To investigate N6AMT2's impact on protein translation, researchers can implement several sophisticated experimental strategies. First, conduct puromycin incorporation assays in cells with normal versus depleted N6AMT2 levels, using N6AMT2 antibodies to confirm knockdown efficiency while measuring nascent protein synthesis rates . Second, perform polysome profiling to assess changes in translating ribosomes following N6AMT2 manipulation, coupled with Western blotting of fractions using N6AMT2 and eEF1A methyl-specific antibodies. Third, employ ribosome profiling (Ribo-seq) in control and N6AMT2-depleted cells to identify specific mRNAs whose translation is most affected by N6AMT2 activity. Fourth, use in vitro translation systems supplemented with recombinant N6AMT2 protein to directly measure its effect on translation elongation rates . Fifth, develop a methyltransferase activity assay using recombinant N6AMT2 (specific activity: 110 pmol/min/mg) and eEF1A substrate to correlate enzymatic activity with translation efficiency . Sixth, conduct proximity ligation assays between N6AMT2 and components of the translation machinery to visualize their interactions in situ. Recent evidence suggesting that eEF1A methylation decreases in aged muscle tissue points to potential age-related dysregulation of translation that may be mediated through changes in N6AMT2 activity .

How can N6AMT2 antibodies be used in tissue-specific methylation studies?

N6AMT2 antibodies provide valuable tools for investigating tissue-specific methylation patterns and their biological significance. For immunohistochemistry applications, researchers should optimize fixation protocols (typically 4% paraformaldehyde for 10 minutes) and antigen retrieval methods to maximize signal while preserving tissue morphology . Tissue microarrays can be employed to simultaneously screen multiple tissues for N6AMT2 expression and localization. For comparative studies between healthy and diseased tissues, dual staining with N6AMT2 antibodies and methyl-specific eEF1A antibodies can reveal correlations between methyltransferase expression and substrate modification . In aging studies, quantitative immunohistochemistry using N6AMT2 antibodies on young versus aged tissue samples can help validate the observation that eEF1A methylation decreases in aged muscle . For mechanistic investigations, laser capture microdissection of specific cell types followed by Western blotting with N6AMT2 antibodies can determine cell-specific expression patterns within heterogeneous tissues. Research suggests that N6AMT2 antibodies are effective in multiple tissue types including colon, skeletal muscle, and pancreatic samples, making them versatile tools for comparative tissue studies . Proper controls are essential, including peptide competition assays and tissues from N6AMT2 knockout models when available.

What are the optimal Western blot protocols for N6AMT2 antibodies?

For optimal Western blot detection of N6AMT2, researchers should implement the following protocol refinements. Sample preparation should include protease inhibitors to prevent degradation of the approximately 31 kDa N6AMT2 protein, and phosphatase inhibitors if investigating potential regulatory phosphorylation. For gel electrophoresis, 12-15% polyacrylamide gels provide optimal resolution for this molecular weight range. Transfer to PVDF membranes is recommended over nitrocellulose for stronger protein binding and signal retention. Blocking should be performed with 3-5% BSA in TBST rather than milk, as milk can contain endogenous phosphatases that may interfere with detection . Primary antibody incubation should be conducted at 1:100 to 1:1000 dilution (depending on the specific antibody) overnight at 4°C . Washing steps should be extended (5 × 5 minutes) to minimize background without compromising specific signal. HRP-conjugated secondary antibodies are typically used at 1:5000 dilution for 1 hour at room temperature. For enhanced sensitivity, especially when detecting endogenous N6AMT2 in tissues with low expression, consider using chemiluminescent substrates with extended signal duration or fluorescently-labeled secondary antibodies with digital imaging systems. When analyzing bands, the expected molecular weight of N6AMT2 should be confirmed, and recombinant N6AMT2 protein can serve as a positive control .

What are the key considerations for immunofluorescence using N6AMT2 antibodies?

Successful immunofluorescence with N6AMT2 antibodies requires careful optimization of multiple parameters. Cell fixation should be performed with 4% paraformaldehyde for 10 minutes at room temperature, followed by permeabilization with 0.2% Triton X-100 for 2 minutes . Blocking with 3% BSA in PBS for 1 hour at room temperature effectively reduces non-specific binding . N6AMT2 antibodies typically perform optimally at 1:100 dilution, though this may vary by manufacturer and should be empirically determined . Include co-staining with subcellular markers (such as DAPI for nuclei or α-tubulin for cytoskeleton) to precisely localize N6AMT2 and provide structural context . To validate specificity, include N6AMT2 knockdown or knockout cells as negative controls, and consider using cells expressing tagged N6AMT2 (such as N6AMT1-EGFP fusion protein) as positive controls . When investigating potential co-localization with eEF1A or ribosomes, super-resolution microscopy techniques may be necessary to resolve the fine spatial relationships. For quantitative analysis, standardize image acquisition parameters and use automated analysis software to measure signal intensity and co-localization coefficients. Given the potential for cross-reactivity seen with related methyltransferases, peptide competition assays should be performed to confirm antibody specificity in immunofluorescence applications .

What controls should be included when using N6AMT2 antibodies in research?

Rigorous control strategies are essential when working with N6AMT2 antibodies to ensure valid and reproducible results. Primary negative controls should include N6AMT2 knockout or knockdown samples, which have proven invaluable in validating antibody specificity . For knockout validation, CRISPR-Cas9 gene editing can generate complete N6AMT2 deletion cell lines such as the U2OS ΔN6AMT1#1 model described in related methyltransferase studies . Positive controls should include samples with confirmed N6AMT2 overexpression, ideally with an orthogonal tag that can be detected independently. For antibody specificity controls, pre-incubation with the immunizing peptide should abolish specific signal in all applications. Technical controls should include secondary-only controls to assess non-specific binding of detection antibodies. When performing knockdown studies, include controls for off-target effects by using multiple siRNA/shRNA sequences targeting different regions of N6AMT2 mRNA. To control for potential cross-reactivity with other methyltransferases, particularly those in the same family, parallel detection with antibodies against related proteins can identify any overlapping signals . For tissue studies, include both positive control tissues (with known N6AMT2 expression) and negative control tissues (with absent or low expression) to establish the dynamic range of detection.

What experimental data supports the role of N6AMT2 in eEF1A methylation?

Strong experimental evidence establishes N6AMT2 (EEF1AKMT1) as a key regulator of eEF1A methylation. Biochemical characterization has demonstrated that N6AMT2 functions as a protein N-lysine methyltransferase that specifically catalyzes the trimethylation of EEF1A at lysine-79 . Recombinant N6AMT2 protein exhibits enzymatic activity of approximately 110 pmol/min/mg in methyltransferase assays . Knockdown experiments have revealed that depletion of N6AMT2 leads to reduced methylation at its target site, confirming its direct role in eEF1A modification . Interestingly, these studies have also uncovered evidence of crosstalk between different methylation sites on eEF1A; N6AMT2 depletion not only affects its primary target (Lys-79) but also impacts eEF1AK36me3 levels . This suggests a complex regulatory network where different methylation events may influence each other, potentially through conformational changes in the eEF1A protein that affect accessibility of other lysine residues to their respective methyltransferases. The development of methyl-specific antibodies against various eEF1A methylation sites has been instrumental in revealing these relationships, allowing researchers to monitor multiple modification states simultaneously and track changes in response to N6AMT2 manipulation .

How does N6AMT2 activity differ between normal and disease states?

While the search results don't provide direct evidence about N6AMT2 activity in disease states, several inferences can be made based on its function and related research. As a methyltransferase that modifies eEF1A, N6AMT2 likely influences protein translation rates, which are frequently dysregulated in various pathological conditions . Given that eEF1A1 expression is increased in cancer cells and eEF1A2 is commonly reexpressed in cancer cells, the methylation status of these translation factors—regulated by N6AMT2—may play a role in malignant transformation or progression . The observation that eEF1A methylation decreases in aged muscle tissue suggests potential implications for age-related disorders and sarcopenia . To investigate these relationships, researchers could use N6AMT2 antibodies for immunohistochemical analysis of disease tissues compared to matched normal samples, such as the pancreatic ductal adenocarcinoma tumor samples mentioned in the search results . Quantitative analysis of N6AMT2 expression and activity across different cancer types, neurodegenerative disorders, and metabolic diseases would help establish whether this methyltransferase represents a potential biomarker or therapeutic target. Additionally, given the role of protein synthesis in stress responses, N6AMT2-mediated eEF1A methylation might be altered under cellular stress conditions that are common in disease states.

What are common issues when using N6AMT2 antibodies and how can they be resolved?

Researchers working with N6AMT2 antibodies may encounter several common technical challenges. First, weak or absent signal in Western blots can occur due to low endogenous expression of N6AMT2 in certain cell types or tissues. This can be addressed by loading more protein (50-100 μg), using more sensitive detection systems, or concentrating proteins through immunoprecipitation before Western blotting . Second, multiple bands or unexpected molecular weight patterns may indicate potential degradation, post-translational modifications, or cross-reactivity with related proteins. To resolve this, use freshly prepared samples with protease inhibitors, compare results with recombinant N6AMT2 protein as a positive control, and validate with knockout/knockdown samples . Third, high background in immunofluorescence or immunohistochemistry applications can obscure specific staining. Improve signal-to-noise ratio by optimizing antibody dilution (typically starting at 1:100), extending blocking time with 3% BSA, and increasing wash duration and frequency . Fourth, inconsistent results between experiments may reflect batch-to-batch variability in polyclonal antibodies. Maintain consistent experimental conditions and consider purchasing larger amounts of a single lot for long-term studies . Fifth, discrepancies between different detection methods (e.g., positive Western blot but negative immunofluorescence) could indicate conformation-dependent epitope recognition. Try multiple antibodies targeting different regions of N6AMT2 or consider alternative fixation and permeabilization protocols that better preserve the epitope structure .

How can researchers improve reproducibility in N6AMT2 antibody experiments?

Enhancing reproducibility in N6AMT2 antibody experiments requires systematic attention to multiple experimental variables. First, maintain detailed records of antibody information including catalog number, lot number, host species, clonality, and immunogen sequence for each experiment . Second, standardize sample preparation protocols, including consistent cell lysis buffers, protein quantification methods, and storage conditions to minimize variability in protein conformation and post-translational modifications. Third, implement quantitative controls in each experiment, such as loading controls for Western blots and internal calibration standards for quantitative immunofluorescence . Fourth, blind sample identity during analysis to prevent unconscious bias in interpretation of results. Fifth, establish clear criteria for positive and negative results before beginning experiments, based on validated positive and negative controls . Sixth, perform statistical power calculations to determine appropriate sample sizes for detecting biologically meaningful differences. Seventh, consider the lessons from cross-reactivity studies of related antibodies, such as the N6AMT1 antibodies that recognized Aurora Kinase A, and implement rigorous specificity testing . Eighth, share detailed protocols including antibody dilutions, incubation times, and buffer compositions in publications to enable other researchers to replicate findings. Finally, validate key findings with orthogonal methods that don't rely on antibody recognition, such as mass spectrometry or activity-based assays for N6AMT2 function .

What factors affect sensitivity and specificity when detecting N6AMT2 in different sample types?

Multiple factors can significantly impact the sensitivity and specificity of N6AMT2 detection across different sample types. For protein lysates, the extraction method critically affects antibody performance; stronger detergents (like SDS) increase protein extraction efficiency but may denature epitopes recognized by conformation-dependent antibodies . Sample processing conditions including heating duration, reducing agent concentration, and storage time can alter protein structure and potentially mask or expose the epitope recognized by N6AMT2 antibodies. In fixed tissue samples, the fixation method and duration profoundly influence antibody binding; paraformaldehyde fixation for 10 minutes generally preserves N6AMT2 epitopes while maintaining cellular architecture . For frozen tissues, the freeze-thaw cycle can damage protein structure, potentially affecting antibody recognition. The abundance of N6AMT2 varies across tissue types, necessitating optimization of antibody concentration for each application; skeletal muscle, colon, and pancreatic tissues have demonstrated detectible levels in previous studies . Post-translational modifications of N6AMT2 itself may differ between tissues or disease states, potentially affecting epitope availability. Background autofluorescence is tissue-specific (particularly high in liver, kidney, and adipose tissue) and can interfere with immunofluorescence detection of N6AMT2. Finally, cross-reactivity risks vary between applications; Western blots separate proteins by size, reducing misidentification, while immunohistochemistry relies solely on spatial distribution and staining intensity, increasing the importance of specificity controls .