NNMT Antibody

What are the recommended applications and dilutions for NNMT antibodies?

NNMT antibodies have been validated across multiple experimental applications with specific optimal dilution ranges for each technique. Based on extensive testing, the following applications and dilutions are recommended:

It's important to note that optimal dilutions may be sample-dependent, and researchers should titrate antibodies in their specific experimental systems . When performing Western blot analysis, NNMT is typically observed at its predicted molecular weight of 30 kDa across various cell types including hepatic, gastric, and osteosarcoma cell lines .

What cell and tissue types are most suitable for NNMT antibody validation?

Multiple cell lines and tissue types have been successfully used to validate NNMT antibodies, providing researchers with reliable positive controls:

Cell lines with confirmed NNMT expression:

• HepG2 cells (human liver cancer)

• L02 cells (human liver)

• U-2 OS cells (human bone osteosarcoma)

• SK-MEL-30 cells (human cutaneous melanoma)

• SiHa cells (human cervical carcinoma)

• A549 cells (human lung adenocarcinoma)

Tissue types with confirmed NNMT expression:

• Human, mouse, and rat liver tissues

• Human gastric tissues (normal and cancerous)

• Human kidney tissue

• Human pancreatic tissue

• Human skin tissue

For negative controls, NNMT knockout HeLa cells have been proven effective, showing complete loss of signal compared to wild-type HeLa cells . For researchers establishing new experimental systems, these validated samples provide essential benchmarks for antibody performance assessment .

What sample preparation methods ensure optimal NNMT detection in Western blotting?

Successful NNMT detection by Western blotting requires careful attention to sample preparation:

• Protein Extraction: Use RIPA buffer supplemented with 1% v/v protease inhibitor cocktail and 1% v/v phosphatase inhibitor cocktail to maintain protein integrity during extraction .

• Protein Quantification: Standardize protein loading at 20 μg per lane for consistent results across experiments .

• Gel Conditions: Use 10-12% SDS-PAGE gels for optimal resolution of the 30 kDa NNMT protein .

• Transfer Parameters: Transfer to PVDF membranes has been validated for NNMT detection; ensure complete transfer of proteins in the 30 kDa range .

• Antibody Incubation: For primary antibodies, overnight incubation at 4°C provides optimal results at the recommended dilutions. Follow with appropriate secondary antibodies (mouse or rabbit IgG-HRP conjugate) for 1 hour at room temperature .

• Controls: Include both positive controls (cell lines known to express NNMT, such as HepG2) and negative controls (NNMT knockout cell lines when available). Use β-Actin or GAPDH as loading controls .

These methodological details are critical for generating reproducible and reliable Western blot results when detecting NNMT in experimental samples.

How can researchers validate NNMT antibody specificity?

Rigorous validation of antibody specificity is essential for reliable NNMT research. Multiple complementary approaches should be employed:

• Genetic Validation Models:

  • Test antibody reactivity in NNMT knockout cell lines (as demonstrated with NNMT knockout HeLa cells)

  • Validate in NNMT knockdown models using targeted shRNA approaches (three validated shRNA sequences have been reported: NM_006169.1-330s1c1, NM_006169.1-164s1c1, and NM_006169.1-448s1c1)

  • Compare signal in wild-type vs. overexpression models to confirm proportional detection

• Multiple Application Testing:

  • Confirm consistent detection across different applications (WB, IHC, IF)

  • Verify appropriate molecular weight (30 kDa) in Western blotting

• Correlation with Alternative Methods:

  • Compare antibody-based detection with RNA-seq data for NNMT expression

  • Correlate protein detection with enzymatic activity measured by LC-MS/MS quantification of nicotinamide and methyl nicotinamide

• Cross-Antibody Validation:

  • Compare results using antibodies targeting different NNMT epitopes

  • Test both monoclonal and polyclonal antibodies when available

These validation strategies ensure that experimental observations genuinely reflect NNMT biology rather than non-specific antibody interactions.

What are the optimal conditions for NNMT immunohistochemical staining?

Successful immunohistochemical detection of NNMT requires optimization of several key parameters:

• Antigen Retrieval: The recommended method is TE buffer at pH 9.0, though citrate buffer at pH 6.0 has also been used as an alternative . The choice between these methods may depend on specific tissue fixation protocols and should be empirically determined for each experimental system.

• Antibody Dilution: For optimal signal-to-noise ratio, a dilution range of 1:400-1:1600 is recommended for most NNMT antibodies in IHC applications .

• Tissue-Specific Considerations: NNMT expression has been successfully visualized in multiple human tissues including liver, kidney, pancreas, and skin using immunohistochemistry at 1/500 antibody dilution .

• Controls: Include positive control tissues with known NNMT expression (such as liver) and negative controls (either NNMT-negative tissues or primary antibody omission controls) .

• Detection Systems: Both chromogenic and fluorescent detection systems have been validated for NNMT visualization, with the choice depending on research requirements for multiplexing or quantification .

For researchers investigating NNMT in cancer tissues, it's important to note that differential expression patterns have been observed between normal and cancerous samples, particularly in gastric cancer where NNMT shows significant upregulation .

How does NNMT antibody performance vary across different cancer models?

NNMT antibodies have been extensively tested across various cancer models, revealing important differences in expression patterns and post-translational modifications:

• Gastric Cancer:

  • Western blotting and 2-DE analyses reveal significantly higher NNMT expression (5.7-fold increase) in gastric cancer tissues compared to normal tissues (P<0.001)

  • Notably, while normal gastric tissues show a single NNMT spot in 2-DE analysis, cancer tissues display 4-5 spots, including additional acidic and basic forms, suggesting cancer-specific post-translational modifications

• Glioblastoma:

  • NNMT expression influences tumor growth rates in U87 glioblastoma models

  • NNMT knockdown cells form tumors significantly slower than wild-type cells (p=3.93E-05), with corresponding reductions in proliferation and colony formation capacity

• Detection Consistency:

  • Across multiple cancer cell lines (U-2 OS, SK-MEL-30, HepG2, SiHa), NNMT antibodies consistently detect the expected 30 kDa band in Western blot analyses

  • Immunohistochemical staining patterns vary by tissue type but maintain specificity when validated antibodies are used at recommended dilutions

These findings highlight the importance of selecting appropriate cancer models when studying NNMT, as expression levels and post-translational modifications may significantly impact antibody performance and experimental outcomes.

How can researchers design functional studies to assess NNMT's role in cancer biology?

Based on established protocols, several experimental approaches are recommended for investigating NNMT's functional significance:

• Isogenic Cell Line Development:

  • Create NNMT knockdown models using validated shRNA sequences (NM_006169.1-330s1c1, NM_006169.1-164s1c1, NM_006169.1-448s1c1)

  • Generate NNMT overexpression models using commercially available true-ORF NNMT human cDNA clones in appropriate expression vectors

  • Validate expression changes by Western blotting with antibody dilutions of 1:1000-1:8000

• Functional Assays:

  • Assess proliferation using MTT assays (NNMT knockdown shows decreased proliferation)

  • Evaluate colony formation capacity (significant reduction observed in NNMT knockdown models)

  • Measure treatment sensitivity by combining radiation therapy with or without chemotherapeutic agents like temozolomide

• In Vivo Tumor Models:

  • Implant isogenic cell lines (knockdown, wild-type, overexpression) in appropriate animal models

  • For intracranial models, NOD-SCID mice have been successfully used with U87 glioblastoma isogenic lines

  • Monitor tumor growth rates and survival outcomes over time

• Molecular Pathway Analysis:

  • Assess key signaling proteins including P-p44/42 MAPK, P-Akt, PP2A-C, and other targets known to be affected by NNMT modulation

  • Investigate apoptotic markers including PARP, Cleaved PARP, Caspase-3, and Cleaved Caspase-3

• Metabolic Analysis:

  • Measure NNMT enzymatic activity by quantifying nicotinamide and methyl nicotinamide concentrations using LC-MS/MS

  • Correlate metabolic changes with phenotypic outcomes in your model system

These experimental approaches provide a comprehensive framework for investigating NNMT's functional role in cancer biology and its potential as a therapeutic target.

What methodological considerations are important when analyzing NNMT in clinical samples?

When investigating NNMT in clinical samples, several methodological considerations are crucial for generating reliable results:

• Sample Collection and Processing:

  • Paired normal and tumor tissue samples provide the most informative comparative analysis, as demonstrated in gastric cancer studies

  • Rapid preservation is critical as NNMT protein may be subject to degradation or modification during processing

• Analytical Techniques:

  • Two-dimensional electrophoresis (2-DE) has proven valuable for detecting post-translational modifications of NNMT in cancer tissues

  • Western blotting using validated antibodies at appropriate dilutions (1:1000-1:8000) provides reliable quantification

  • Immunohistochemistry at 1:400-1:1600 dilution allows visualization of spatial expression patterns

• Modified Protein Analysis:

  • The observation of additional acidic and basic forms of NNMT in cancer tissues suggests important post-translational modifications

  • Multiple spots (4-5) detected in cancer tissues compared to a single spot in normal tissues may represent cancer-specific protein modifications

• Validation Approaches:

  • Monoclonal antibodies with sensitivity down to 10 ng have been developed specifically for clinical sample analysis

  • When analyzing large cohorts, standardized protocols and scoring systems are essential for consistent results

• Clinical Correlation:

  • Correlate NNMT expression patterns with clinical parameters including tumor stage, treatment response, and patient outcomes

  • Consider both expression level and post-translational modifications as potentially distinct biomarkers

These methodological considerations are essential for accurately characterizing NNMT in clinical samples and for establishing its potential as a diagnostic or prognostic biomarker.

How can researchers address common challenges in NNMT Western blotting?

When encountering difficulties with NNMT Western blotting, consider the following troubleshooting approaches:

• Weak or Absent Signal:

  • Verify NNMT expression in your sample type (confirmed expression in liver, gastric tissues, HepG2, L02, U-2 OS, and SK-MEL-30 cells)

  • Optimize antibody concentration (test a range from 1:1000-1:8000)

  • Increase protein loading (20 μg per lane has been validated)

  • Extend primary antibody incubation time to overnight at 4°C

  • Ensure complete protein transfer from gel to membrane, particularly for the 30 kDa region

• Multiple Bands or Non-specific Binding:

  • Validate antibody specificity using NNMT knockout control samples

  • Increase blocking stringency (5% non-fat milk or BSA)

  • Optimize washing conditions (increase duration or number of washes)

  • Try alternative validated NNMT antibodies targeting different epitopes

• Inconsistent Results:

  • Standardize protein extraction method (RIPA buffer with protease and phosphatase inhibitors)

  • Ensure consistent sample handling and storage conditions

  • Validate loading controls (β-Actin or GAPDH) for equal sample loading

  • Consider the impact of post-translational modifications, which may alter antibody recognition

• Unexpected Molecular Weight:

  • NNMT should appear at approximately 30 kDa

  • In some cancer tissues, multiple NNMT spots with varying isoelectric points may represent post-translational modifications

  • Verify gel percentage (10-12% SDS-PAGE gels are optimal for resolving NNMT)

By systematically addressing these common challenges, researchers can optimize NNMT Western blotting for reliable and reproducible results.

What controls are essential for validating NNMT antibody experiments?

A comprehensive set of controls is crucial for ensuring the validity of NNMT antibody experiments:

• Positive Controls:

  • Cell lines with confirmed NNMT expression:

    • HepG2 cells (human liver cancer)

    • L02 cells (human liver)

    • U-2 OS cells (human bone osteosarcoma)

    • SK-MEL-30 cells (human melanoma)

  • Tissue samples with validated expression:

    • Human, mouse, and rat liver tissues

    • Human gastric cancer tissues

• Negative Controls:

  • NNMT knockout cell lines (NNMT knockout HeLa cells show complete signal loss)

  • NNMT knockdown models created using validated shRNA constructs

  • Primary antibody omission controls for immunostaining applications

• Technical Controls:

  • Loading controls for Western blotting (GAPDH, β-Actin)

  • Isotype controls for immunohistochemistry and flow cytometry

  • Secondary antibody-only controls to assess non-specific binding

• Expression Validation Controls:

  • Correlation with RNA-seq data for NNMT expression

  • Enzymatic activity measurement (nicotinamide and methyl nicotinamide quantification)

  • Multiple antibodies targeting different NNMT epitopes

• Application-Specific Controls:

  • For 2-DE analysis: Internal standard proteins for alignment

  • For IHC: Positive and negative tissue controls processed simultaneously

  • For functional studies: Complete isogenic series (knockdown, wild-type, overexpression)

Implementing these controls ensures that experimental observations genuinely reflect NNMT biology rather than technical artifacts or non-specific interactions.

How does NNMT silencing impact cellular signaling pathways?

NNMT silencing has significant effects on multiple cellular signaling pathways, providing insight into its potential as a therapeutic target:

• Tumor Suppressor Activation:

  • NNMT silencing activates the tumor suppressor protein phosphatase 2A (PP2A)

  • This activation involves changes in protein methylation status, with implications for downstream signaling events

• MAPK Pathway Modulation:

  • Western blot analysis using phospho-specific antibodies reveals altered MAPK signaling in NNMT-modulated cells

  • P-p44/42 MAPK levels change in response to NNMT silencing

• PI3K/Akt Pathway Effects:

  • NNMT knockdown influences Akt phosphorylation (Ser473)

  • This pathway modulation may contribute to the observed changes in proliferation and survival capacity

• Stress Response Pathway Engagement:

  • ASK1 and p38 signaling components are affected by NNMT modulation

  • P-p38 levels can be monitored to assess stress response activation

• Apoptotic Pathway Sensitivity:

  • NNMT knockdown cells show altered levels of apoptotic markers including PARP, Cleaved PARP, Caspase-3, and Cleaved Caspase-3

  • These changes correlate with increased sensitivity to treatments like radiation therapy

The comprehensive analysis of these signaling changes requires careful Western blotting with validated antibodies for each pathway component, following the sample preparation and detection methods described earlier.

What is the significance of NNMT post-translational modifications in cancer tissues?

Research using 2-DE and Western blot analysis has revealed important differences in NNMT post-translational modifications between normal and cancer tissues:

• Pattern Differences:

  • In gastric ulcer tissues, NNMT appears as a single spot on 2-DE gels

  • In gastric cancer tissues, NNMT presents as 4-5 distinct spots, indicating significant post-translational modifications

• Novel Isoforms:

  • Cancer tissues display additional acidic and basic forms of NNMT not detected in normal tissues

  • These modifications appear to be cancer-specific, suggesting potential roles in pathogenesis

• Detection Methodology:

  • Western blot analysis using monoclonal antibodies with detection limits down to 10 ng can visualize these modified forms

  • The antibody specificity allows detection of the various post-translationally modified variants

• Potential Functional Implications:

  • These modifications may alter NNMT enzymatic activity, protein-protein interactions, or subcellular localization

  • They could represent potential cancer-specific biomarkers with diagnostic or prognostic value

• Research Approaches:

  • To investigate these modifications, researchers should combine 2-DE with Western blotting using validated NNMT antibodies

  • Mass spectrometry analysis of the different spots can identify the specific nature of these modifications

Understanding these cancer-specific post-translational modifications may provide new insights into NNMT's role in cancer biology and offer potential targeted therapeutic approaches.