NMI Antibody

What is NMI and why is it significant in cancer research?

NMI (N-myc interactor) is a protein that interacts with the oncogene Myc family members NMYC and CMYC, as well as other transcription factors containing Zip, HLH, or HLH-Zip motifs. Its significance in cancer research stems from its interaction with critical signaling pathways and its differential expression patterns. NMI interacts with most STAT proteins (excluding STAT2) and enhances STAT-mediated transcription responses to cytokines like IL2 and IFN-gamma . Furthermore, NMI has been identified as an ARF-interacting protein that protects ARF from ULF-mediated ubiquitin degradation, revealing its involvement in stress response pathways . Expression analysis shows NMI has low expression in most human tissues except brain but demonstrates high expression in myeloid leukemias, suggesting a potential role in hematological malignancies .

What are the key applications for NMI antibodies in experimental research?

NMI antibodies serve multiple experimental purposes across various research methodologies. They are validated for Western blotting (WB), immunohistochemistry (IHC), immunofluorescence (IF), immunocytochemistry (ICC), and flow cytometry applications . In Western blotting, NMI antibodies detect a specific band at approximately 35 kDa, corresponding to the calculated molecular weight of the protein . For immunohistochemistry and immunocytochemistry, they enable visualization of NMI expression patterns in tissues and cellular compartments, which is particularly valuable for studying translocation phenomena such as the stress-induced nuclear migration of NMI . Flow cytometry applications allow quantitative assessment of NMI expression levels in specific cell populations, facilitating more comprehensive analyses of expression patterns across different cell types or experimental conditions .

How do researcher-grade NMI antibodies differ from standard antibodies?

Research-grade NMI antibodies like the Picoband series are characterized by enhanced quality parameters that address the specific needs of investigators conducting sensitive experiments. These premium antibodies demonstrate higher affinity, improved specificity, and reduced background noise compared to standard options . The Picoband NMI antibody undergoes rigorous validation across multiple applications and sample types, ensuring reliable performance in diverse experimental contexts. This validation includes testing against various human cell lines (K562, Caco-2, A431, PC-3, 293T) and tissues (placenta, colon cancer, lung cancer, mammary cancer), providing researchers with confidence in cross-application reliability . The enhanced signal-to-noise ratio of these antibodies is particularly beneficial for detecting low-abundance proteins or for applications requiring precise quantification of subtle expression differences.

What are the optimal protocol modifications for detecting NMI in different tissue types?

Detection of NMI across diverse tissue types requires specific methodological adjustments to account for tissue-specific characteristics and protein expression levels. For formalin-fixed paraffin-embedded (FFPE) tissue sections, heat-mediated antigen retrieval in EDTA buffer (pH 8.0) has been validated as an effective approach for unmasking NMI epitopes . The blocking step with 10% goat serum is critical for minimizing non-specific binding, particularly in tissues with high endogenous immunoglobulin content . Antibody concentration should be optimized based on tissue type, with 1μg/ml being effective for various cancer tissues and placenta . For tissues with known low NMI expression (most fetal and adult tissues except brain), extended primary antibody incubation times (overnight at 4°C) and signal amplification systems like Strepavidin-Biotin-Complex (SABC) with DAB chromogen development can enhance detection sensitivity .

How should researchers optimize Western blot conditions for NMI detection in cell lysates?

Optimization of Western blotting for NMI detection requires attention to several critical parameters. Sample preparation should include 50μg of protein per lane under reducing conditions, with separation performed on 5-20% SDS-PAGE gradients run at 70V (stacking gel) followed by 90V (resolving gel) for 2-3 hours to achieve optimal protein separation . After electrophoresis, proteins should be transferred to nitrocellulose membranes at 150mA for 50-90 minutes, followed by blocking with 5% non-fat milk in TBS for 1.5 hours at room temperature . The rabbit anti-NMI antibody performs optimally at a concentration of 0.5 μg/mL when incubated overnight at 4°C . Signal development using enhanced chemiluminescent detection provides the sensitivity required to visualize the specific 35 kDa NMI band across various human cell lysates. For cell lines with potentially lower NMI expression, increasing the primary antibody concentration and extending the signal development time may improve detection without compromising specificity .

What controls are essential for validating NMI antibody specificity in experimental systems?

Comprehensive validation of NMI antibody specificity requires implementation of multiple control strategies. Positive controls should include tissues or cell lines with known NMI expression (such as myeloid leukemia cell lines or brain tissue) . Negative controls should include tissues with minimal NMI expression, as well as technical controls where primary antibody is omitted or replaced with non-specific IgG from the same species . For flow cytometry applications, isotype control antibodies (rabbit IgG at equivalent concentrations) and unlabelled samples should be processed in parallel to establish baseline fluorescence and non-specific binding parameters . Cross-reactivity assessment is particularly important, especially when working with non-human samples, as the antibody's reactivity may vary between species . When investigating protein-protein interactions involving NMI, reciprocal co-immunoprecipitation experiments are essential to confirm the specificity of observed interactions, as demonstrated in studies of NMI-ARF and NMI-ULF interactions .

How can NMI antibodies be used to study protein-protein interactions in cellular stress responses?

Investigating NMI's role in cellular stress responses requires sophisticated application of NMI antibodies in combination with other experimental techniques. Coimmunoprecipitation (CoIP) assays using anti-NMI antibodies can detect stress-induced changes in NMI's interaction network, as demonstrated in studies identifying NMI-ARF and NMI-ULF interactions . For optimal results, CoIP protocols should include crosslinking steps to stabilize transient interactions that may occur during stress responses. Complementing CoIP experiments with in vitro binding assays using purified components (His-NMI and GST-ARF) provides direct evidence of protein-protein interactions independent of cellular contexts . For examining stress-induced subcellular translocation of NMI, immunofluorescence microscopy using anti-NMI antibodies in combination with nuclear markers enables visualization of NMI's movement from cytoplasm to nucleus following stress stimuli . Time-course experiments tracking NMI localization after stress induction can reveal the kinetics of this translocation process and its correlation with downstream effects on ARF stabilization and stress response pathway activation .

What approaches can resolve contradictory NMI expression data in different experimental systems?

Resolving conflicting NMI expression data across experimental systems requires systematic methodological considerations. Researchers should first verify antibody performance across all systems being compared through validation experiments using positive and negative controls appropriate for each experimental context . Cross-platform normalization strategies should be employed when comparing data from different detection methods (e.g., Western blot, IHC, flow cytometry). For quantitative comparisons, researchers should generate standard curves using recombinant NMI protein to calibrate detection across different experimental batches and platforms . Post-translational modifications of NMI might affect antibody recognition in different contexts, so complementary detection approaches targeting different epitopes can help resolve discrepancies. The table below outlines a systematic approach for troubleshooting contradictory expression data:

How can researchers effectively study NMI's role in the transcription-independent regulation of ARF?

Investigating NMI's function in transcription-independent ARF regulation requires sophisticated experimental designs. The protein half-life assay is a fundamental technique in this context, where cycloheximide (CHX) is used to block protein synthesis, allowing observation of ARF degradation kinetics with and without NMI expression . Western blotting at defined time points post-CHX treatment (0, 3, 6, 9 hours) enables calculation of ARF half-life under different experimental conditions . To establish that NMI's effect on ARF is post-transcriptional, quantitative real-time RT-PCR analysis should be performed to confirm that ARF mRNA levels remain unchanged despite altered protein levels . For mechanistic studies examining how NMI protects ARF from ULF-mediated degradation, in vitro ubiquitination assays can be performed, comparing ubiquitination levels of ARF in the presence and absence of NMI . The interaction network should be confirmed through a series of coimmunoprecipitation experiments testing all combinations of ARF, NMI, and ULF to establish the molecular basis of this regulatory mechanism .

What are the most effective strategies for increasing signal-to-noise ratio in NMI immunostaining?

Enhancing the signal-to-noise ratio in NMI immunostaining requires a multifaceted approach targeting various aspects of the protocol. Optimization of antigen retrieval is paramount, with EDTA buffer (pH 8.0) demonstrating superior performance compared to citrate buffer for NMI epitope exposure in FFPE tissues . Extended blocking steps (10% goat serum for at least 1 hour) significantly reduce background staining, particularly in tissues with high endogenous peroxidase activity . For chromogenic detection methods, using diaminobenzidine (DAB) as the substrate in conjunction with Strepavidin-Biotin-Complex (SABC) amplification systems enhances sensitivity while maintaining low background . Temperature control during antibody incubation is critical; primary antibody incubation at 4°C overnight followed by secondary antibody incubation at 37°C for 30 minutes has been validated to maximize specific binding while minimizing non-specific interactions . For fluorescent detection, careful selection of fluorophores with minimal spectral overlap and inclusion of autofluorescence quenching steps significantly improves visual clarity, particularly in tissues with high endogenous fluorescence .

How should researchers address cross-reactivity concerns when applying NMI antibodies to non-human samples?

Addressing cross-reactivity concerns when using human-targeted NMI antibodies for non-human samples requires careful validation and control experiments. While human NMI shares 64% amino acid sequence identity with mouse NMI, the reactivity of antibodies across species boundaries cannot be automatically assumed . Researchers should perform preliminary Western blot experiments using both human and target species samples in parallel to assess cross-reactivity and detection efficiency . If cross-reactivity is observed, epitope mapping can help identify the conserved regions being recognized, providing insights into potential differences in binding affinity between species. When cross-species application is attempted, antibody concentration may need to be adjusted; typically higher concentrations are required for non-validated species applications . For definitive confirmation of specificity in new species, knockdown or knockout controls in the target species are invaluable for distinguishing specific from non-specific signals . The customer inquiry referenced in the search results specifically asked about using the human-reactive anti-NMI antibody for dog tissues, highlighting the common need for cross-species applications in comparative studies .

What are the critical considerations for quantitative analysis of NMI expression across different experimental conditions?

Quantitative analysis of NMI expression requires rigorous methodological standardization to ensure accurate comparisons across experimental conditions. For Western blot quantification, consistent loading controls must be selected based on their stability under the specific experimental conditions being tested . Digital image acquisition parameters should be optimized to ensure signals fall within the linear dynamic range of detection, avoiding both signal saturation and insufficient sensitivity issues . For immunohistochemical quantification, standardized scoring systems should be established, preferably incorporating both staining intensity and percentage of positive cells, as demonstrated in the validation images of NMI expression in different cancer tissues . Flow cytometric analysis offers superior quantitative precision for cellular NMI expression, but requires careful gating strategies and consistent instrument calibration between experimental runs . The table below outlines critical parameters for ensuring quantitative reliability:

How can NMI antibodies be utilized in studies examining stress-induced protein translocation mechanisms?

NMI antibodies offer powerful tools for investigating stress-induced protein translocation phenomena, particularly NMI's movement from cytoplasm to nucleus under stress conditions. Immunofluorescence microscopy using anti-NMI antibodies combined with nuclear counterstains provides visual evidence of this translocation process . For quantitative assessment of translocation dynamics, high-content imaging systems can track the nuclear-to-cytoplasmic ratio of NMI signal intensity across time after stress induction. Subcellular fractionation followed by Western blotting with anti-NMI antibodies offers biochemical confirmation of translocation, providing quantifiable data on the proportion of NMI in different cellular compartments . Chromatin immunoprecipitation (ChIP) assays using anti-NMI antibodies can determine whether nuclear-translocated NMI associates with specific genomic regions, potentially revealing direct transcriptional regulatory functions beyond its role in ARF stabilization . Live-cell imaging approaches using fluorescently tagged NMI constructs, validated against antibody-detected endogenous NMI, can provide real-time visualization of translocation kinetics in response to various stressors, revealing the temporal relationship between NMI movement and downstream pathway activation .

What protocols can be developed for studying NMI's role in STAT-mediated transcription responses?

Investigating NMI's role in augmenting STAT-mediated transcription requires specialized experimental approaches combining NMI antibodies with transcriptional analysis techniques. Chromatin immunoprecipitation (ChIP) assays using anti-NMI antibodies can identify genomic regions where NMI associates with STAT transcription factors following cytokine stimulation . Sequential ChIP (re-ChIP) protocols, first immunoprecipitating with anti-STAT antibodies followed by anti-NMI antibodies, can confirm co-occupancy of both proteins at specific genomic loci. For functional analysis, luciferase reporter assays containing STAT-responsive elements can be used to quantify transcriptional activity in the presence of varying NMI levels, establishing dose-dependent relationships between NMI expression and STAT-mediated transcription . Co-immunoprecipitation experiments using anti-NMI antibodies followed by Western blotting for various STAT proteins can confirm the specific STAT family members interacting with NMI in different cellular contexts and in response to specific cytokines like IL2 and IFN-gamma . RNA-seq analysis comparing transcriptomes of control versus NMI-overexpressing or NMI-depleted cells after cytokine stimulation can identify the global impact of NMI on STAT-regulated gene expression programs .

How can researchers design experiments to explore the therapeutic potential of targeting NMI in cancer?

Designing experiments to investigate NMI as a therapeutic target in cancer requires multifaceted approaches spanning from mechanistic studies to preclinical models. Immunohistochemical analysis using anti-NMI antibodies across tissue microarrays of various cancer types can establish correlations between NMI expression levels and clinical parameters or survival outcomes . The validated detection of NMI in colon, lung, and mammary cancer tissues provides a foundation for such analyses . For mechanistic exploration, genetic manipulation of NMI levels (overexpression, knockdown, knockout) followed by functional assays measuring proliferation, apoptosis, migration, and invasion can establish the phenotypic consequences of NMI modulation in cancer cells . Drug screening platforms can be developed using NMI expression or activity as readouts, employing NMI antibodies in high-throughput immunoassays to identify compounds that modulate NMI levels or disrupt key interactions . In vivo studies using xenograft models with manipulated NMI expression can assess the impact on tumor growth, metastasis, and response to standard therapies . For validating NMI as a biomarker, analysis of circulating NMI in patient samples (using sensitive immunoassays based on validated antibodies) can determine its potential utility for diagnosis, prognosis, or treatment monitoring .