NMT1 Antibody

What is NMT1 and why is it significant in research?

NMT1 (N-myristoyltransferase 1) is an essential enzyme responsible for catalyzing the transfer of myristate from CoA to proteins. It facilitates N-myristoylation, a critical post-translational modification where myristate, a 14-carbon saturated fatty acid, is attached via an amide linkage to the N-terminal glycine residue of numerous cellular and viral proteins . This modification is irreversible and crucial for the proper biological functioning of many myristoylated proteins, including components of signal transduction pathways such as the alpha subunit of G protein GO (GNAO1) . Recent research has shown that NMT1 plays pivotal roles in tumorigenesis, particularly in liver cancer development, making it an important research target for understanding cancer mechanisms .

What are the molecular characteristics of NMT1 protein?

NMT1 protein has the following key characteristics:

• Full Name: N-myristoyltransferase 1 (also known by synonyms including NMT-S, NMT-L)

• Calculated Molecular Weight: 496 amino acids, approximately 57 kDa

• Observed Molecular Weight: 50-60 kDa in experimental conditions

• Gene ID (NCBI): 4836

• UniProt ID: P30419

• Primary Function: Transfer of myristate from CoA to proteins with N-terminal glycine residues

What are the typical applications for NMT1 antibodies in research?

NMT1 antibodies are versatile tools employed in multiple research applications:

The antibodies have demonstrated reactivity with human, mouse, and rat samples, making them suitable for comparative studies across species .

How can researchers evaluate NMT1-dependent protein myristoylation?

Researchers can employ several sophisticated techniques to assess NMT1-dependent protein myristoylation:

• Click Chemistry Assay: This method enables detection of myristoylated proteins by using alkyne-tagged myristoyl-CoA analogs (e.g., Alk12-CoA). The protocol involves:

  • Treating cells with the NMT1/NMT2 inhibitor DDD85646 to control background myristoylation

  • Expression and purification of target proteins

  • In vitro modification with recombinant NMT1 and Alk12-CoA

  • Conjugation of an azide-containing fluorescent dye to the alkyne tag via click chemistry

  • Analysis of labeling by in-gel fluorescence

• Metabolic Labeling: Cells are incubated with azido-myristate, followed by cell lysis, click chemistry reaction with alkyne-TAMRA, and visualization of myristoylated proteins by Western blot using anti-TAMRA antibodies .

• Protein Ligation Assay (PLA): This technique can identify direct interactions between NMT1 and target proteins:

  • Cells are fixed with paraformaldehyde

  • Primary antibodies against NMT1 and the potential substrate protein are applied

  • PLA probes are added, followed by ligase and amplification-polymerase solutions

  • Proximity (< 40 nm) between the two proteins results in detectable fluorescent signals

What approaches can validate the specificity of an NMT1 antibody?

Validating NMT1 antibody specificity is crucial for reliable experimental results. Recommended validation strategies include:

• Genetic Knockdown/Knockout Controls:

  • Use siRNA targeting NMT1 (such as NMT1-1 siRNA) in cell lines like SK-OV-3

  • Compare with control siRNA and NMT2-targeted siRNA to confirm specificity

  • Analyze by Western blot to verify reduced signal corresponding to NMT1 depletion

• Liver-Specific Knockout Model: For in vivo research, liver-conditional NMT1 knockout mice (NMT1 flox/flox: Alb-Cre) provide an excellent system to validate antibody specificity and study NMT1 function:

  • Generate by crossing NMT1 flox/+ mice with Albumin (Alb) Cre mice

  • Confirm genotype via PCR

  • Validate knockout efficiency using the antibody in question

• Multiple Detection Methods: Confirm findings using orthogonal techniques:

  • Compare immunohistochemistry results with Western blot data

  • Validate with immunoprecipitation followed by mass spectrometry

How does NMT1 contribute to liver tumorigenesis?

Recent research has elucidated NMT1's critical role in liver cancer development:

• Expression Pattern: NMT1 shows elevated expression in liver cancer tissues compared to adjacent normal tissues, suggesting its potential as a biomarker .

• Functional Impact: Liver-specific NMT1 knockout mice (NMT1 flox/flox: Alb-Cre) exhibit reduced tumorigenesis in DEN/CCl4-induced liver cancer models, demonstrating that NMT1 is essential for liver tumor development .

• Mechanistic Insights: NMT1 mediates its oncogenic effects through:

  • Regulating post-translational N-myristoylation of key proteins involved in cancer pathways

  • Potentially modifying proteins like AHSG and RPL29 that were identified in proteomics studies

  • Affecting protein localization to membrane surfaces, which is critical for many signaling cascades

• Experimental Approaches: Researchers investigating NMT1 in liver cancer typically employ:

  • Tissue microarrays (TMA) with paired cancer/normal tissues

  • IHC using optimized dilutions (1:500-1:2000) for NMT1 antibodies

  • Parallel reaction monitoring (PRM) to evaluate expression levels

  • Proteomics (iTraq) to identify proteins affected by NMT1 knockdown

What distinguishes NMT1 from NMT2 in experimental design?

Understanding the differences between NMT1 and NMT2 is essential for proper experimental design:

• Enzymatic Activities:

  • Both NMT1 and NMT2 can myristoylate N-terminal glycine residues

  • Remarkably, both enzymes can also myristoylate specific lysine residues, representing a novel type of protein modification

  • In vitro studies using recombinant enzymes show that both can modify substrates like ARF6, including both G2 (glycine) and K3 (lysine) sites

• Experimental Distinction:

  • Use specific antibodies: anti-NMT1 (such as Abcam #ab186123) and anti-NMT2 (Abcam #ab224045)

  • Apply selective knockdown: NMT1-1 siRNA and NMT2-4 siRNA produce distinct effects on protein expression patterns

  • Conditional knockout models: NMT1 flox/flox: Alb-Cre mice provide specific insights into NMT1 function without affecting NMT2

• Functional Redundancy: When designing experiments targeting either enzyme, consider potential compensatory mechanisms between NMT1 and NMT2.

What are optimal storage and handling conditions for NMT1 antibodies?

Proper handling ensures antibody integrity and experimental reproducibility:

How can click chemistry enhance NMT1 substrate identification?

Click chemistry has revolutionized the study of NMT1 and protein myristoylation through:

• Substrate Detection Protocol:

  • Cells are treated with myristic acid analog containing an alkyne group (e.g., YnMyr)

  • After cell lysis, the click chemistry reaction attaches an azide-containing tag to myristoylated proteins

  • The click reaction mixture typically includes CuSO4 (1 mM), TCEP (1 mM), and TBTA (0.1 mM)

  • After protein precipitation with methanol containing EDTA (10 mM), proteins are resuspended in sample buffer

  • Analysis by Western blot using antibodies against the tag (e.g., anti-TAMRA)

• Advantages Over Traditional Methods:

  • Higher sensitivity compared to radioactive labeling

  • Better specificity for identifying true myristoylation substrates

  • Compatibility with proteomics workflows for large-scale substrate identification

  • Ability to visualize myristoylated proteins in fixed cells or tissues

What controls are essential when studying NMT1 in cancer models?

Rigorous experimental controls are crucial for reliable NMT1 research in cancer models:

• Genetic Controls:

  • Include both NMT1 and NMT2 knockdown conditions

  • Use scrambled siRNA or non-targeting CRISPR guides as negative controls

  • In mouse models, compare NMT1 flox/flox: Alb-Cre with NMT1 flox/flox (without Cre) as controls

• Pharmacological Controls:

  • Treat cells with NMT inhibitors (e.g., DDD85646) to control background myristoylation

  • Include dose-response studies to establish inhibitor specificity

  • Compare effects with other lipid modification inhibitors to confirm specificity

• Tissue Controls:

  • Always include paired normal/tumor tissue samples

  • Use tissue microarrays (TMAs) with multiple patient samples to account for heterogeneity

  • Apply standardized antigen retrieval methods (TE buffer pH 9.0 or citrate buffer pH 6.0)

What are the best techniques for studying NMT1 interactions with substrates?

Several techniques can elucidate NMT1-substrate interactions:

• Co-Immunoprecipitation (Co-IP):

  • Lyse cells under conditions that preserve protein-protein interactions

  • Immunoprecipitate with anti-NMT1 antibody

  • Detect potential substrates in the precipitated complex

  • Alternatively, immunoprecipitate candidate substrates and probe for NMT1

• Protein Ligation Assay (PLA):

  • Particularly useful for detecting transient or weak interactions

  • Requires proximity (<40 nm) between proteins

  • Uses antibodies against both proteins of interest (e.g., anti-FLAG for tagged NMT1 and anti-HA for tagged substrate)

  • Provides spatial information about interactions within cells

• In Vitro Myristoylation Assays:

  • Express and purify potential substrate proteins

  • Incubate with recombinant NMT1 and Alk12-CoA

  • Perform click chemistry and visualize by in-gel fluorescence

  • Compare wild-type substrates with mutants (e.g., G2A, K3R) to identify specific myristoylation sites

How can researchers address inconsistent NMT1 antibody staining?

Inconsistent staining is a common challenge when working with NMT1 antibodies. Methodological solutions include:

• Optimization of Antigen Retrieval:

  • For IHC applications, test both TE buffer (pH 9.0) and citrate buffer (pH 6.0)

  • Adjust retrieval time and temperature based on tissue type

  • Consider microwave-based vs. pressure cooker-based retrieval methods

• Antibody Validation:

  • Confirm specificity using positive and negative controls

  • Include NMT1 knockdown/knockout samples as negative controls

  • Test multiple antibody clones if available

• Sample Preparation Considerations:

  • Ensure consistent fixation protocols for tissues

  • For cell lines, standardize confluence and growth conditions

  • Consider cell-specific expression levels when troubleshooting

What are the emerging applications of NMT1 research beyond cancer?

While NMT1's role in cancer is well-documented, research is expanding into other areas:

• Infectious Disease Applications:

  • NMT1 regulates myristoylation of viral proteins

  • Potential therapeutic target for viral infections

  • Can be studied using viral protein constructs and NMT1 inhibitors

• Cellular Signaling Research:

  • NMT1 affects membrane localization of signaling proteins

  • Critical for G-protein function and downstream pathways

  • Can be investigated using signaling-specific reporter assays

• Developmental Biology:

  • Essential for organism viability (insertional mutagenesis of Nmt1 in yeast is lethal)

  • Tissue-specific conditional knockout models provide insights into developmental roles

  • Can be studied using temporal and spatial genetic manipulation approaches

How can proteomics approaches enhance NMT1 substrate identification?

Proteomics offers powerful tools for comprehensive identification of NMT1 substrates:

• iTraq-Based Approaches:

  • Isobaric tags for relative and absolute quantification (iTraq) can identify proteins affected by NMT1 knockdown

  • Compare protein expression profiles between control and NMT1-knockdown samples

  • Analyze data to identify both direct substrates and downstream effectors

• Click Chemistry-Coupled Proteomics:

  • Metabolically label cells with alkyne-myristate

  • Perform click chemistry with azide-biotin

  • Enrich myristoylated proteins using streptavidin

  • Identify enriched proteins by mass spectrometry

  • Compare results with and without NMT1 inhibition or knockdown

• Parallel Reaction Monitoring (PRM):

  • Targeted proteomics approach to evaluate expression of protein modification enzymes

  • Particularly useful for analyzing paired tissue samples

  • Provides quantitative data on NMT1 and related enzymes

By implementing these advanced approaches and considering the methodological details provided, researchers can design robust experiments to investigate NMT1 function and its implications in various biological contexts.