NME1 Antibody

What is NME1 and why is it important in biomedical research?

NME1 (also known as nm23-H1) serves as a metastasis suppressor protein involved in multiple cellular processes. It has significant importance in cancer research due to its metastasis suppression role. Studies have demonstrated that NME1 is upregulated in ductal carcinoma in situ (DCIS) compared to normal breast epithelial tissues, but its levels decrease in microinvasive and invasive components of breast tumor cells . This expression pattern makes NME1 a crucial protein for understanding cancer progression mechanisms, particularly the transition from in situ to invasive disease. Additionally, NME1 functions as a nucleoside diphosphate kinase and has been identified as a coenzyme A (CoA) binding protein that undergoes modification under oxidative stress conditions .

What types of NME1 antibodies are available for research applications?

Researchers have access to several types of NME1 antibodies with validated applications:

• Mouse monoclonal antibodies such as CPTC-NME1-2 (IgG2a isotype), which have been extensively characterized for multiple applications

• Antibody pairs consisting of rabbit polyclonal capture antibodies combined with mouse monoclonal detection antibodies for quantitative assays

• Specialized antibodies validated for specific applications like imaging mass cytometry

Many of these antibodies have been comprehensively validated across multiple tissue types, including both normal tissues (liver, bone marrow, spleen, prostate, colon, pancreas, breast, lung, testis, endometrium, and appendix) and cancer tissues (breast, colon, ovarian, lung, and prostate) .

What experimental applications can NME1 antibodies be used for?

NME1 antibodies have been validated for numerous research applications:

For IHC applications, the CPTC-NME1-2 antibody shows specific cytoplasmic staining in multiple tissue types, including various cancer tissues. This antibody has also been successfully employed in the NCI60 Cell Line Array with variable expression patterns across different cell lines .

How should I optimize NME1 antibody protocols for immunohistochemistry?

For optimal immunohistochemistry results with NME1 antibodies, follow these methodological guidelines:

• Start with a validated dilution range - 1:50 has been successfully used for antibodies like CPTC-NME1-2 on tissue microarrays

• Perform antigen retrieval optimization for your specific tissue type (heat-induced epitope retrieval in citrate buffer pH 6.0 is often effective)

• Include appropriate positive control tissues (colon, lung cancer, breast tissues have shown reliable staining)

• Run a dilution series (1:25, 1:50, 1:100, 1:200) to determine optimal concentration for your specific tissues

• For multiplex applications like imaging mass cytometry, a 1:100 dilution of 0.5mg/mL stock has shown positive results across multiple tissue types

The staining pattern should be predominantly cytoplasmic, though some nuclear staining may be observed depending on the cell type and physiological state. Always evaluate staining specificity by comparing with the known biology of NME1 and including appropriate negative controls.

What are the optimal methods for detecting NME1-protein interactions?

To study NME1-protein interactions, several approaches have proven effective:

• Pull-down assays: As demonstrated in studies of NME1-CaMKII interactions, use 6xHis-tagged proteins immobilized on nickel-nitrilotriacetic acid agarose resin and incubate with cell/tissue lysates containing NME1 or with purified NME1 protein . After washing, elute bound proteins and analyze by SDS-PAGE followed by immunoblotting with anti-NME1 antibodies.

• Co-immunoprecipitation: Use anti-NME1 antibodies to immunoprecipitate NME1 along with its binding partners. For example:

  • Prepare cell lysates under non-denaturing conditions

  • Pre-clear with protein A/G beads

  • Incubate with anti-NME1 antibody overnight at 4°C

  • Capture complexes with protein A/G beads

  • Analyze by western blotting for potential interaction partners

• Proximity ligation assays: For in situ detection of protein interactions in fixed cells or tissues, using pairs of antibodies against NME1 and potential interacting proteins.

This approach has successfully identified interactions between NME1 and proteins such as CaMKII, revealing that NME1 directly interacts with CaMKII and modulates its activity in a concentration-dependent manner .

How can I address cross-reactivity between NME1 and NME2 in my experiments?

Cross-reactivity between NME1 and NME2 is an important consideration due to their high sequence homology. Research-validated approaches to address this include:

• Antibody selection: Review cross-reactivity data for your selected antibody. For example, cross-reactivity data is available for antibodies like CPTC-NME1-2

• Validation controls:

  • Include recombinant NME1 and NME2 proteins as positive and specificity controls

  • For critical experiments, include NME1 knockout/knockdown samples

  • Use western blotting to distinguish between NME1 and NME2 based on their slight molecular weight differences

• Multiple antibody approach: Validate key findings with multiple antibodies targeting different epitopes

• Isoform-specific detection: When possible, target epitopes in regions that differ between NME1 and NME2

• Biochemical separation: Use techniques like 2D gel electrophoresis to separate the isoforms before immunodetection

This systematic approach ensures reliable discrimination between these highly similar proteins and prevents misinterpretation of experimental results.

How can I use NME1 antibodies to study its concentration-dependent effects on CaMKII activity?

To investigate NME1's biphasic effect on CaMKII activity (enhancement at nanomolar concentrations and inhibition at micromolar concentrations), researchers can employ these methodological approaches:

• In vitro kinase assays:

  • Use purified components (recombinant NME1 and CaMKII)

  • Test a concentration range of NME1 (20 nM to 10 μM) with constant CaMKII

  • Monitor activity via:

    • Pyruvate kinase/lactate dehydrogenase (PK/LDH) coupled assays

    • Direct kinase assays with radiolabeled ATP

    • Luminescence-based endpoint assays that quantify ATP

• Autophosphorylation studies:

  • Incubate CaMKII with Ca²⁺/calmodulin in the presence of varying NME1 concentrations

  • Detect CaMKII autophosphorylation by immunoblotting with phospho-specific antibodies

  • Compare autophosphorylation levels across NME1 concentration range

• Cellular studies:

  • Generate cell lines with titratable NME1 expression

  • Measure endogenous CaMKII activity using phospho-specific antibodies

  • Correlate CaMKII activity with NME1 expression levels

These approaches have revealed that NME1 significantly enhances CaMKII activity at 500 nM concentration but strongly inhibits activity at 4 μM concentration. This inhibition appears independent of calmodulin concentration, suggesting NME1 does not act by sequestering calmodulin .

What techniques can I use to study NME1 CoAlation under oxidative stress?

To investigate NME1 CoAlation (covalent modification by Coenzyme A) under oxidative stress conditions, employ the following research-validated approaches:

• Recombinant protein studies:

  • Express and purify wild-type NME1 and C109A mutant (deficient in CoAlation at this site)

  • Perform in vitro CoAlation reactions under oxidizing conditions

  • Analyze by western blotting with anti-CoA and anti-NME1 antibodies

• Cellular oxidative stress models:

  • Transfect cells with His-tagged NME1 constructs (wild-type and C109A mutant)

  • Treat with oxidizing agents (diamide at 500 μM for 30 min or H₂O₂ at 100 μM to 2.5 mM for 30 min)

  • For metabolic stress, culture in glucose- and pyruvate-free media for 20 hours

  • Harvest cells with NEM to prevent post-lysis thiol modifications

  • Perform His-tag pull-downs using Ni-NTA resin

  • Analyze by western blotting for CoAlation status

• Mass spectrometry confirmation:

  • Perform affinity purification of NME1 from treated cells

  • Digest with trypsin and analyze by LC-MS/MS

  • Identify the CoAlated peptide containing Cys109

  • Compare spectral counts between oxidizing conditions and controls

This approach has successfully identified NME1 as a major CoA binding protein in cultured cells and tissues, with specific CoAlation occurring at Cys109 under oxidative stress conditions .

How can I investigate NME1's role in breast cancer invasion?

To study NME1's function in regulating the invasive transition in breast cancer, employ these methodological approaches:

• Expression analysis in clinical samples:

  • Perform immunohistochemistry with anti-NME1 antibodies on breast cancer tissue microarrays containing matched DCIS and invasive components

  • Quantify NME1 expression using digital image analysis

  • Correlate expression with clinical parameters and outcomes

• Mechanistic studies:

  • Conduct dual immunofluorescence for NME1 and MT1-MMP to analyze their anti-correlation in invasive components

  • Use triple staining to visualize NME1, MT1-MMP, and dynamin-2 in clathrin-coated vesicles

  • Perform co-immunoprecipitation studies to confirm protein interactions

• Functional analysis in cell models:

  • Generate NME1 knockout cell lines using CRISPR-Cas9

  • Measure MT1-MMP surface levels by flow cytometry or cell surface biotinylation

  • Assess ECM degradation using fluorescent gelatin degradation assays

  • Perform invasion assays using Matrigel-coated transwell chambers

• In vivo validation:

  • Utilize the intraductal xenograft model with control and NME1-knockout cells

  • Monitor invasive progression by immunohistochemistry

  • Quantify metastatic spread to lymph nodes and distant organs

Research has shown that NME1 levels drop during the progression from DCIS to invasive breast cancer, and this downregulation enhances MT1-MMP surface levels by inhibiting its endocytic clearance, thereby promoting ECM degradation and invasion .

What essential controls should be included in NME1 antibody experiments?

For rigorous NME1 antibody experiments, incorporate these controls:

• Positive controls:

  • Cell lines or tissues with known NME1 expression (e.g., specific breast cancer cell lines)

  • Recombinant NME1 protein for western blotting

  • FFPE tissues validated for positive staining (colon, liver, specific cancer types)

• Negative controls:

  • Primary antibody omission

  • NME1 knockout or knockdown samples

  • Isotype controls matching the primary antibody's isotype (e.g., mouse IgG2a for CPTC-NME1-2)

  • Tissues known to lack NME1 expression

• Specificity controls:

  • Pre-absorption with recombinant NME1 protein

  • Validation with multiple antibodies targeting different epitopes

  • Peptide competition assays

• Cross-reactivity controls:

  • Recombinant NME1 and NME2 proteins to assess specificity

  • Western blot analysis to distinguish based on molecular weight

These controls ensure reliable and reproducible results across different experimental applications and prevent misinterpretation due to non-specific binding or false-positive/negative outcomes.

How do I troubleshoot weak or non-specific NME1 antibody staining?

When encountering staining issues with NME1 antibodies, use these empirically-validated troubleshooting approaches:

For weak staining:

• Optimize antibody concentration - validated dilutions for IHC include 1:50 for CPTC-NME1-2

• Extend primary antibody incubation (overnight at 4°C instead of 1 hour at room temperature)

• Improve antigen retrieval:

  • Test different buffers (citrate pH 6.0 vs. EDTA pH 9.0)

  • Extend retrieval time or increase temperature

• Use signal amplification systems (tyramide signal amplification, polymer-based detection)

• Check tissue fixation protocols (overfixation can mask epitopes)

For non-specific staining:

• Optimize blocking:

  • Increase blocking duration (1 hour minimum)

  • Try different blocking agents (BSA, normal serum from detection antibody species)

• Dilute antibody further

• Perform more stringent washing (increased duration, higher salt concentration)

• Try alternative antibodies targeting different epitopes

• For IHC with avidin-biotin systems, include biotin blocking step

Following these methodological adjustments has proven effective in optimizing NME1 detection across multiple tissue types and experimental conditions .

How can I validate NME1 antibody specificity for critical experiments?

For critical experiments requiring high confidence in antibody specificity, implement this comprehensive validation strategy:

• Biochemical validation:

  • Perform western blot analysis to confirm detection of a band at the correct molecular weight (~17-20 kDa for NME1)

  • Run recombinant NME1 as positive control

  • Include NME2 to assess cross-reactivity

• Genetic validation:

  • Compare samples from NME1 knockout models with wild-type

  • Analyze siRNA/shRNA knockdown samples alongside non-targeting controls

  • Use graded knockdown to establish correlation between signal and expression level

• Epitope validation:

  • Perform peptide competition assays

  • Use multiple antibodies targeting different epitopes and compare staining patterns

  • For custom antibodies, validate with epitope-mutated constructs

• Orthogonal validation:

  • Compare protein detection with mRNA expression (RNA-seq, qPCR, in situ hybridization)

  • Verify subcellular localization patterns match known biology

  • Perform mass spectrometry on immunoprecipitated samples

This multi-layered validation strategy ensures that experimental findings truly reflect NME1 biology rather than artifacts of antibody cross-reactivity or non-specific binding.

How can I implement imaging mass cytometry with NME1 antibodies?

Imaging mass cytometry (IMC) enables highly multiplexed protein detection in tissues. For NME1 analysis via IMC, follow this methodological workflow:

• Antibody preparation:

  • Metal-label the NME1 antibody (CPTC-NME1-2 has been validated for IMC)

  • Titrate to determine optimal concentration (typically 1:100 dilution of 0.5mg/mL stock)

  • Validate specificity using positive and negative controls

• Sample preparation:

  • Prepare FFPE tissue sections (4-5 μm thickness)

  • Perform deparaffinization and antigen retrieval

  • Block with BSA/normal serum

• Multiplex staining:

  • Combine metal-labeled NME1 antibody with other markers of interest

  • Include markers for tissue architecture (e.g., E-cadherin, cytokeratins)

  • Add immune cell markers if studying tumor microenvironment

• Data acquisition and analysis:

  • Acquire data using a Hyperion Imaging System or equivalent

  • Process using specialized software (Visiopharm, HALO)

  • Perform neighborhood analysis to identify cellular interactions

This approach has been successfully applied to multiple tissue types including normal colon, liver, bone marrow, spleen, and various cancer tissues, allowing visualization of NME1 expression in spatial context .

How can I investigate the relationship between NME1 and CaMKII in cancer progression?

To study the functional relationship between NME1 and CaMKII in cancer, implement this research strategy based on their concentration-dependent interaction :

• Expression correlation analysis:

  • Perform dual immunofluorescence or sequential IHC on cancer tissue microarrays

  • Quantify NME1 and phospho-CaMKII (activated form) levels

  • Analyze correlation at different cancer stages and in different tissue compartments

• Functional studies in cell models:

  • Generate cell lines with:

    • NME1 knockdown/knockout

    • NME1 overexpression (wild-type and binding mutants)

    • Inducible/titratable NME1 expression

  • Measure:

    • CaMKII activity via phospho-specific antibodies

    • Cancer-related phenotypes (migration, invasion, proliferation)

    • Rescue experiments with CaMKII inhibitors

• Mechanistic investigation:

  • Study NME1-CaMKII complex formation at different NME1 concentrations

  • Analyze downstream CaMKII substrates in relation to NME1 levels

  • Examine how oxidative stress affects the NME1-CaMKII axis

This approach leverages the finding that NME1 enhances CaMKII activity at nanomolar concentrations but inhibits it at micromolar concentrations , which may be relevant to its concentration-dependent effects in different cancer contexts.

What approaches can I use to study NME1 CoAlation in relation to its metastasis suppressor function?

To investigate how CoAlation of NME1 at Cys109 affects its metastasis suppressor function, employ this comprehensive research strategy:

• Cellular model development:

  • Generate stable cell lines expressing:

    • Wild-type NME1

    • C109A mutant (CoAlation-deficient)

    • C109D mutant (phosphomimetic to simulate modification)

  • Validate expression by western blotting with NME1 antibodies

• Functional phenotype assessment:

  • Invasion assays (Boyden chamber, 3D matrix)

  • Migration assays (wound healing, single-cell tracking)

  • Adhesion and colony formation assays

  • Compare phenotypes under normal and oxidative stress conditions

• Biochemical characterization:

  • Measure nucleoside diphosphate kinase activity of wild-type vs. mutant NME1

  • Analyze protein-protein interactions under oxidative stress

  • Assess ability to modulate CaMKII activity

• In vivo studies:

  • Orthotopic xenograft models with wild-type or C109A mutant NME1 cells

  • Tail vein injection metastasis assays

  • Analyze primary tumors and metastases by IHC with anti-NME1 antibodies

• Translational relevance:

  • Examine CoAlation status of NME1 in patient samples using specialized antibodies

  • Correlate with oxidative stress markers and metastatic outcomes

This approach connects the biochemical modification of NME1 by CoA with its established role in suppressing metastasis , potentially revealing new therapeutic strategies targeting this regulatory mechanism.