NME2 Antibody

What is NME2 and what cellular functions does it regulate?

NME2 (Nucleoside diphosphate kinase B) is an enzyme encoded by the NME2 gene in humans. It exists as a hexamer composed of 'A' (encoded by NME1) and 'B' (encoded by NME2) isoforms . The protein plays critical roles in several cellular processes, including the phosphorylation of nucleoside diphosphates, negative regulation of Rho activity through interaction with AKAP13/LBC, and transcriptional activation of the MYC gene . Additionally, NME2 has been shown to bind DNA non-specifically and is involved in reducing proliferation, migration, and invasion of cancer cells, particularly in gastric cancer models .

What are the commonly used applications for NME2 antibodies?

NME2 antibodies have been validated for multiple research applications including:

• Western Blot (WB): Typically used at dilutions of 1:500-1:1000 to detect NME2 at its expected molecular weight of approximately 17 kDa .

• Immunohistochemistry (IHC): Used at dilutions of 1:50-1:500 to detect NME2 in tissue sections, particularly effective in kidney tissues .

• Immunofluorescence (IF)/Immunocytochemistry (ICC): Applied at dilutions of 1:50-1:500 to visualize NME2 localization in cells such as HEK-293 .

• Flow cytometry: Used in conjunction with cell cycle analysis to study NME2's role in proliferation .

The choice of application depends on the specific research question, with western blotting being most commonly used for quantitative analysis of expression levels, while immunofluorescence provides insights into subcellular localization .

Which species reactivity is confirmed for commonly available NME2 antibodies?

Commercial NME2 antibodies have been validated for reactivity across multiple species:

When selecting an antibody for cross-species studies, polyclonal antibodies often provide broader reactivity, while monoclonal antibodies typically offer higher specificity for a single epitope .

How can researchers distinguish between NME1, NME2, and NME1-NME2 fusion proteins in experimental systems?

Distinguishing between these closely related proteins requires careful antibody selection and experimental design:

For specific detection of NME2 only, researchers should use antibodies that target unique epitopes not present in NME1. The MA1-185 monoclonal antibody is designed specifically for human NME2 detection and does not cross-react with NME1 .

For detection of NME1-NME2 fusion proteins, which result from naturally-occurring transcripts encoding a fusion protein with sequences from both genes, antibodies like NBP3-05576 that target the fusion protein should be employed . This antibody was raised against a recombinant fusion protein containing amino acids 1-267 of human NME1-NME2 (NP_001018146.1) .

To confirm specificity in experimental systems, researchers should:

• Perform parallel western blots with antibodies specific to each protein

• Include appropriate positive controls of known molecular weights

• Consider using gene knockdown (siRNA) experiments as negative controls

• For ultimate verification, implement mass spectrometry to identify the exact protein species present .

What methodologies are most effective for studying NME2's role in cancer cell metastasis?

Based on published research methodologies, a comprehensive approach to studying NME2's role in cancer metastasis should include:

• Cell migration assays:

  • Wound-healing assay: Create scratches in confluent cell monolayers expressing different levels of NME2 and monitor closure rates over 24-48 hours .

  • Transwell migration assay: Quantitatively measure cell migration through membranes, comparing cells with normal vs. altered NME2 expression .

• Invasion assays:

  • Collagen-coated Transwell chambers: Assess the ability of cells to invade through extracellular matrix by counting cells that transmigrate through collagen-coated membranes .

• Cell cycle analysis:

  • Flow cytometry: Fix cells with 70% ethanol, treat with RNase A, stain with propidium iodide, and analyze DNA content to determine if NME2 affects cell cycle progression .

• Protein localization:

  • Immunofluorescence: Use anti-NME2 antibodies (1:100 dilution) followed by fluorescent-conjugated secondary antibodies to visualize subcellular localization, with DAPI counterstaining for nuclei .

• In vivo metastasis models:

  • Orthotopic implantation of cells with different NME2 expression levels can provide more physiologically relevant data on metastatic potential.

These methodologies should be used comparatively between control cells and those with NME2 overexpression or knockdown to establish cause-effect relationships .

What are the known technical challenges in detecting phosphorylation states of NME2?

Detecting phosphorylation states of NME2 presents several technical challenges:

• Autophosphorylation mechanism: NME2 functions as a nucleoside diphosphate kinase that transfers phosphate groups and can undergo autophosphorylation at histidine residues. These His-phosphorylation states are notoriously unstable in acidic conditions commonly used in protein extraction buffers .

• Phospho-specific antibody limitations: While phospho-specific antibodies for serine, threonine, and tyrosine phosphorylation sites are common, antibodies specific for histidine phosphorylation (relevant for NME2) are rare and technically challenging to develop.

• Buffer considerations: Researchers should use neutral to slightly basic buffers (pH 7.5-8.0) during protein extraction and handling to preserve histidine phosphorylation states.

• Detection methods: Mass spectrometry-based approaches are currently the most reliable for detecting and characterizing NME2 phosphorylation states, though they require specialized equipment and expertise.

• Controls: Appropriate controls are essential, including dephosphorylated samples (through phosphatase treatment) and samples with known phosphorylation states.

For accurate phosphorylation analysis, a combination of radioisotope labeling, phospho-enrichment techniques, and mass spectrometry currently offers the most comprehensive approach .

What are the optimal protocols for immunofluorescence staining using NME2 antibodies?

The following optimized protocol for immunofluorescence staining with NME2 antibodies is based on published methodologies:

• Cell preparation:

  • Plate cells on glass coverslips and allow growth to desired confluency (typically 60-80%) .

  • Rinse twice with ice-cold PBS .

  • Fix in 4% paraformaldehyde in PBS for 15 minutes at room temperature .

  • Permeabilize with 0.5% Triton X-100 for 10 minutes .

  • Block non-specific binding with 1% bovine serum albumin (BSA) in PBS for 30 minutes .

• Antibody incubation:

  • Primary antibody: Dilute NME2 antibody in PBS containing 1% BSA. Recommended dilutions range from 1:50 to 1:500, with 1:100 being commonly used .

  • Incubate with primary antibody for 24 hours at 4°C or 1-2 hours at room temperature in a humidified chamber .

  • Wash 3 times with PBS, 5 minutes each.

  • Secondary antibody: Incubate with appropriate fluorophore-conjugated secondary antibody (1:100-1:500) for 1 hour at room temperature in the dark .

  • Wash 3 times with PBS, 5 minutes each.

• Nuclear counterstaining and mounting:

  • Counterstain nuclei with DAPI (1:100 in PBS) for 10 minutes at room temperature .

  • Wash 2 times with PBS.

  • Mount coverslips on slides using anti-fade mounting medium.

  • Seal edges with nail polish and store at 4°C protected from light.

• Optimization tips:

  • Always include a negative control (omitting primary antibody) to assess background fluorescence.

  • For cells with low NME2 expression, signal amplification systems may be needed.

  • When studying NME2 localization in relation to other proteins, dual immunofluorescence can be performed with appropriate antibody combinations .

How should researchers optimize western blot protocols for NME2 detection?

Optimizing western blot protocols for NME2 detection requires attention to several key parameters:

• Sample preparation:

  • Extract proteins using RIPA buffer or NP-40 lysis buffer with protease inhibitors.

  • For phosphorylation studies, include phosphatase inhibitors and maintain neutral pH.

  • Load 25-30 μg of total protein per lane as determined from published protocols .

• Gel electrophoresis and transfer:

  • Use 12-15% SDS-PAGE gels to properly resolve the 17 kDa NME2 protein .

  • Transfer to PVDF or nitrocellulose membranes at 100V for 1 hour or 30V overnight at 4°C.

  • Confirm transfer efficiency with Ponceau S staining.

• Antibody incubation:

  • Block membranes with 3-5% non-fat dry milk or BSA in TBST for 1 hour .

  • Primary antibody dilutions:

    • MA1-185: Successfully tested for western blot (dilution not specified)

    • NBP3-05576: 1:3000 dilution recommended

    • 20493-1-AP: 1:500-1:1000 dilution recommended

  • Incubate with primary antibody overnight at 4°C.

  • Wash 3-5 times with TBST.

  • Incubate with HRP-conjugated secondary antibody (typically 1:10000) for 1 hour at room temperature .

  • Wash 3-5 times with TBST.

• Detection:

  • Use ECL substrate for detection, with exposure times of approximately 30 seconds for strong signals .

  • For weak signals, consider enhanced ECL substrates or longer exposure times.

• Controls and validation:

  • Positive controls: Jurkat cells, mouse kidney tissue, and mouse liver tissue have been validated for NME2 expression .

  • Negative controls: Consider using NME2 knockdown cells.

  • Expected band size: Approximately 17 kDa for NME2 and 22 kDa for NME1-NME2 fusion protein .

What statistical approaches are recommended for analyzing NME2 expression data in cancer studies?

Based on published methodologies, the following statistical approaches are recommended for analyzing NME2 expression data in cancer studies:

• Correlation with clinical parameters:

  • Fisher's exact test or chi-square test: Appropriate for defining the relationship between NME2 expression and categorical pathological characteristics of cancer tissue .

  • Pearson correlation analysis: Suitable for assessing correlation between NME2 expression levels and continuous variables such as degree of differentiation and metastatic potential of cancer cells .

• Comparing experimental groups:

  • One-way ANOVA: Recommended for group comparison among parental cells and cells transfected with either a vehicle vector or a human NME2 cDNA .

  • Post-hoc tests (e.g., Tukey's test): Should be applied following ANOVA when comparing multiple groups.

  • Student's t-test: Appropriate when comparing only two groups (e.g., high vs. low NME2 expression).

• Survival analysis:

  • Kaplan-Meier method with log-rank test: Ideal for analyzing the relationship between NME2 expression levels and patient survival.

  • Cox proportional hazards regression: Useful for multivariate analysis to determine if NME2 is an independent prognostic factor.

• Visualization and reporting:

  • Data should be expressed as mean ± SEM (Standard Error of Mean) as standard practice .

  • GraphPad Prism or similar software is recommended for generating publication-quality graphs.

  • SPSS (version 20.0 or later) has been successfully used for statistical analyses in published NME2 research .

• Sample size considerations:

  • Power analysis should be performed prior to experiments to determine appropriate sample sizes.

  • For cell-based assays, a minimum of three independent experiments with technical triplicates is recommended.

A p-value < 0.05 is typically considered statistically significant in NME2 research publications .

How should researchers interpret conflicting results between NME2 expression levels and cancer cell behavior?

When faced with conflicting results regarding NME2 expression and cancer cell behavior, researchers should consider several factors:

• Cell type-specific effects: NME2 may have opposite effects in different cancer types or even subtypes within the same cancer. For example, while NME2 has been shown to reduce proliferation, migration, and invasion in gastric cancer models , its effects might differ in other cancer types. Researchers should carefully compare their cell models with those in published studies.

• NME2 isoforms and modifications: Consider whether discrepancies might be due to:

  • Different NME1/NME2 ratios in the hexameric complex

  • Presence of NME1-NME2 fusion proteins

  • Post-translational modifications affecting function

  • Nuclear versus cytoplasmic localization of NME2

• Experimental methodologies:

  • Different approaches (wound healing vs. Transwell) may yield different results .

  • Validate findings using complementary methods. For migration studies, both wound-healing and Transwell assays should be performed as the former is semi-quantitative while the latter provides more quantitative data .

• Microenvironmental factors:

  • Consider whether conflicting results might be due to differences in culture conditions, matrix components, or paracrine signaling.

  • 2D versus 3D culture systems may reveal different aspects of NME2 function.

• Statistical rigor:

  • Ensure proper statistical analysis, including adequate sample sizes and appropriate tests .

  • Consider whether outliers were appropriately handled and if distributions were normal.

When publishing conflicting findings, researchers should directly address discrepancies with previous literature, proposing testable hypotheses that might explain the differences observed.

What are the key considerations when comparing human and mouse NME2 antibody data?

When comparing human and mouse NME2 antibody data, researchers should consider several important factors:

• Sequence homology and cross-reactivity:

  • While human and mouse NME2 share high sequence homology, species-specific differences exist that may affect antibody binding.

  • Some antibodies like NBP3-05576 and 20493-1-AP have confirmed cross-reactivity with both human and mouse NME2 , but binding efficiency may differ.

  • Researchers should verify cross-reactivity experimentally rather than relying solely on manufacturer claims.

• Molecular weight differences:

  • The observed molecular weight for human NME2 is approximately 17 kDa .

  • Mouse NME2 may migrate slightly differently on SDS-PAGE gels due to sequence differences.

  • Corresponding UniProt IDs for reference: Human (P22392), Mouse (Q01768), Rat (P19804) .

• Expression patterns and tissue distribution:

  • Expression levels of NME2 vary between species and tissues.

  • Mouse kidney and liver tissues have been validated as positive controls for NME2 expression .

  • Different antibody dilutions may be required for optimal detection in different species.

• Experimental validation:

  • Always include appropriate species-specific positive controls.

  • Consider using knockout/knockdown models as negative controls to confirm specificity.

  • When possible, validate key findings with multiple antibodies targeting different epitopes.

• Functional conservation:

  • Despite high sequence conservation, regulatory mechanisms governing NME2 function may differ between species.

  • Context-dependent interactions with other proteins may vary across species.

Researchers should explicitly state which species was used in each experiment and avoid extrapolating findings across species without proper validation .

How can researchers accurately assess NME2's role in cell proliferation versus metastasis?

Accurately distinguishing NME2's effects on proliferation versus metastasis requires careful experimental design:

• Separating proliferation from migration/invasion effects:

  • Time-controlled experiments: Short-term migration/invasion assays (24 hours) minimize the confounding effect of proliferation differences .

  • Proliferation normalization: For longer experiments, normalize migration/invasion data to proliferation rates measured in parallel.

  • Proliferation inhibitors: Consider using mitomycin C or other proliferation inhibitors during migration assays to eliminate proliferation as a variable.

• Comprehensive proliferation assessment:

  • Cell cycle analysis: Flow cytometry with propidium iodide staining provides detailed information on cell cycle distribution, revealing whether NME2 affects specific phases .

  • Proliferation markers: Immunostaining for Ki-67 or BrdU incorporation assays can complement direct cell counting methods.

  • Real-time monitoring: Systems like IncuCyte or xCELLigence allow continuous monitoring of proliferation without endpoint limitations.

• Metastasis-specific assays:

  • Invasion through extracellular matrix: Collagen-coated Transwell assays specifically assess invasive capacity .

  • 3D organotypic models: These provide more physiologically relevant environments for studying invasion.

  • In vivo models: Orthotopic implantation followed by assessment of distant metastasis provides the most definitive evidence of metastatic capacity.

• Molecular pathway analysis:

  • Rho GTPase activity: Since NME2 negatively controls Rho activity , direct measurement of Rho activation (e.g., GLISA) can help distinguish cytoskeletal effects relevant to migration from proliferative effects.

  • MYC regulation: As NME2 acts as a transcriptional activator of MYC , assessing MYC-dependent transcription can help attribute proliferative effects.

• Multi-parameter analysis:

  • Perform correlation analyses between NME2 expression, proliferation markers, and metastasis markers in the same samples.

  • Use multivariate statistical approaches to determine independent contributions of NME2 to each process .

By systematically addressing these aspects, researchers can more accurately delineate the specific contributions of NME2 to proliferation versus metastatic processes .

What emerging technologies might enhance NME2 antibody-based research?

Several emerging technologies offer promising avenues for advancing NME2 antibody-based research:

• Proximity labeling techniques:

  • BioID or APEX2 fusion with NME2 could reveal proximal interacting partners in living cells, providing insights into context-specific protein interactions.

  • These approaches could help identify novel binding partners that mediate NME2's effects on proliferation, migration, and invasion.

• Super-resolution microscopy:

  • Techniques such as STORM, PALM, or STED microscopy used with highly specific NME2 antibodies could reveal subcellular localization with unprecedented precision.

  • This could help resolve questions about nuclear versus cytoplasmic functions of NME2.

• Single-cell analysis:

  • Single-cell proteomics combined with NME2 antibodies could reveal cell-to-cell variability in expression and correlation with metastatic potential.

  • Single-cell RNA-seq paired with antibody-based protein detection (CITE-seq) could link transcriptional programs with NME2 protein levels.

• Antibody engineering:

  • Development of phospho-specific antibodies for histidine phosphorylation would greatly enhance studies of NME2 enzymatic activity.

  • Nanobodies or single-domain antibodies against NME2 could enable live-cell imaging and functional perturbation.

• Spatial transcriptomics and proteomics:

  • Technologies that preserve spatial information while measuring NME2 expression could reveal important insights about its role in tumor heterogeneity and the tumor microenvironment.

These technologies could address current limitations in understanding NME2's multifaceted roles in cancer and other biological processes, potentially leading to new therapeutic strategies targeting NME2-dependent pathways .

How might NME2 antibodies contribute to therapeutic development in cancer?

NME2 antibodies have significant potential to contribute to cancer therapeutic development in several ways:

• Biomarker development and patient stratification:

  • Standardized immunohistochemical protocols using validated NME2 antibodies could help stratify patients based on expression levels .

  • This stratification could predict metastatic potential and guide treatment decisions, particularly in gastric cancer where NME2's role in reducing proliferation, migration, and invasion has been demonstrated .

• Functional antibodies as therapeutics:

  • Antibodies that can enhance NME2's metastasis-suppressive functions could potentially be developed into therapeutic agents.

  • Intracellular antibody delivery technologies (such as cell-penetrating antibodies) could potentially target intracellular NME2 to modulate its function.

• Drug discovery and validation:

  • High-throughput screening assays using NME2 antibodies could identify compounds that modulate NME2 expression or activity.

  • Antibody-based assays can serve as critical validation tools for measuring cellular responses to potential therapeutics targeting NME2-related pathways.

• Monitoring treatment response:

  • NME2 antibodies could be used to monitor changes in expression or localization during treatment, potentially serving as pharmacodynamic markers.

  • These antibodies could help determine whether therapeutic interventions successfully modulate NME2-dependent pathways.

• Combination therapy approaches:

  • Understanding NME2's interactions with other proteins through co-immunoprecipitation with specific antibodies could reveal potential synergistic targets for combination therapy.

  • This is particularly relevant given NME2's known interactions with Rho pathways and MYC transcriptional regulation .