nat8l Antibody

What is NAT8L and what are its primary biological functions?

NAT8L (N-acetyltransferase 8-like) is a member of the N-acyltransferase superfamily that catalyzes the synthesis of N-acetylaspartate (NAA) from L-aspartate and acetyl-CoA. This enzyme plays multiple critical roles in cellular metabolism:

• In neural tissue: Synthesizes NAA, a major brain-specific metabolite essential for myelination

• In metabolism: Promotes dopamine uptake by regulating TNF-alpha expression and attenuates methamphetamine-induced inhibition of dopamine uptake

• In adipose tissue: Influences lipid turnover, mitochondrial biogenesis, and energy metabolism in brown adipocytes

The protein contains a conserved sequence characteristic of the GCN5 or NAT superfamily of N-acetyltransferases and functions as a single-pass membrane protein . While NAA is traditionally considered brain-specific, recent research has identified significant NAT8L expression in adipose tissues, suggesting broader metabolic roles .

What are the recommended applications for NAT8L antibodies?

Based on extensive validation data, NAT8L antibodies have demonstrated effectiveness in multiple applications:

Most commercially available NAT8L antibodies are polyclonal rabbit antibodies that target specific epitopes in the protein sequence. When selecting an antibody, researchers should consider both the application needs and the target species, as reactivity varies between products .

How should NAT8L antibodies be stored and handled to maintain optimal reactivity?

For maximum stability and performance of NAT8L antibodies, researchers should follow these evidence-based handling protocols:

• Storage temperature: Store at -20°C for long-term stability

• Buffer conditions: Most NAT8L antibodies are supplied in PBS with 0.02-0.09% sodium azide and may contain 0.5% BSA or 50% glycerol for stabilization

• Aliquoting: Divide into small aliquots upon receipt to avoid repeated freeze-thaw cycles which can degrade antibody performance

• Stability: When properly stored, NAT8L antibodies typically remain stable for one year after shipment

• Working dilutions: Prepare fresh working dilutions on the day of the experiment for optimal binding specificity

It's worth noting that 20 μL size preparations often contain 0.1% BSA as an additional stabilizing agent .

What is the optimal protocol for Western blotting with NAT8L antibodies?

For successful Western blot detection of NAT8L, researchers should consider these methodological details:

• Sample preparation considerations:

  • Brain tissue samples (mouse/rat) and HUVEC cells have been validated as positive controls

  • The calculated molecular weight of NAT8L is approximately 33 kDa, but the observed molecular weight is typically 45-48 kDa on SDS-PAGE

• Protocol optimization:

  • Begin with recommended dilutions (1:500-1:2000) and titrate for your specific system

  • Protein transfer efficiency is crucial as NAT8L can show variable migration patterns

  • Include appropriate positive controls (brain tissue) and negative controls

  • For detection, both chemiluminescence and fluorescent secondary antibodies have been successfully employed

• Troubleshooting tips:

  • If nonspecific bands appear, increase blocking time or antibody dilution

  • If signal is weak, consider longer incubation times with primary antibody (overnight at 4°C)

  • NAT8L expression varies significantly between tissues; adjust loading accordingly

This methodological approach has been validated in multiple publications referencing NAT8L antibodies .

How can researchers validate NAT8L antibody specificity in experimental systems?

To ensure experimental rigor, researchers should employ the following validation strategies for NAT8L antibodies:

• siRNA-mediated knockdown:

  • Use siRNA targeting NAT8L (validated in H1299 and HCC4017 cell lines)

  • The most efficient protein-level knockdown has been achieved with specific siRNA sequences (si(NAT8L)#1)

  • Western blotting to confirm protein reduction (typically 68-72% knockdown efficiency)

• Functional validation:

  • Measure intracellular NAA levels after NAT8L knockdown (expected 72% reduction)

  • Assess metabolic impacts on precursors (pyruvate, aspartate) which should remain largely unchanged

  • Monitor extracellular NAA levels which should decrease selectively compared to other secreted metabolites

• Immunohistochemical validation:

  • Compare staining patterns in tissues known to express NAT8L (brain, lung cancer) versus negative controls

  • Use peptide competition assays to confirm binding specificity to the immunizing peptide

These approaches provide complementary evidence for antibody specificity beyond standard Western blot controls .

How does NAT8L expression correlate with cancer progression in non-small cell lung cancer (NSCLC)?

The relationship between NAT8L expression and NSCLC provides important insights for cancer researchers:

• Expression patterns:

  • NAT8L is selectively overexpressed in approximately 40-44% of lung adenocarcinomas and 38-40% of squamous cell carcinomas

  • Expression analysis of The Cancer Genome Atlas (TCGA) data revealed significant elevation of NAT8L expression (>2-fold, p<0.01) in tumors compared to patient-matched non-malignant lung tissues

  • NAT8L protein expression is consistently higher in NSCLC cell lines compared to immortalized normal lung epithelium (HBEC30KT, HBEC34KT)

• Functional significance:

  • NAT8L catalyzes the production of N-acetylaspartate (NAA), which is detectable in 10/11 NSCLC tumor samples (4.5-56.7 μM) but absent in non-malignant lung tissues

  • Intracellular NAA levels correlate reasonably well with secreted NAA levels (R²=0.62) in NSCLC cell lines

  • NAA may serve as a potential circulating biomarker, with 46% of NSCLC patients aged ≤55 years showing elevated NAA blood levels (>60 nM threshold)

• Experimental approaches:

  • RNA-seq analysis of tumor vs. normal tissue

  • Western blot quantification of NAT8L protein expression

  • Metabolomic profiling to detect NAA levels in tissues and blood

  • siRNA-mediated knockdown to assess functional dependencies

These findings suggest NAT8L could be a valuable cancer-specific marker and potential therapeutic target in NSCLC .

What experimental approaches can detect NAT8L-dependent NAA production in cell culture?

Researchers investigating NAT8L function can employ these validated methodological approaches:

• Metabolite measurement techniques:

  • Gas chromatography-mass spectrometry (GC-MS) for quantification of intracellular NAA levels

  • Liquid chromatography-mass spectrometry for analysis of secreted NAA in conditioned media

  • Stable isotope labeling to track carbon flow from glucose/glutamine to NAA via NAT8L activity

• Experimental manipulations:

  • siRNA-mediated NAT8L knockdown followed by metabolite profiling

  • Nutrient availability modulation (especially glutamine) which impacts NAA biosynthesis

  • Overexpression studies to assess dose-dependent effects on NAA production

• Controls and validation:

  • Measure related metabolites (pyruvate, aspartate, lactate, alanine) to confirm specificity

  • Include cell lines with known NAT8L expression levels as positive and negative controls

  • Validate with multiple independent siRNA sequences to rule out off-target effects

These approaches have successfully demonstrated that NAT8L is functionally involved in NAA production in various cell types, with NAA serving as a direct and specific readout of NAT8L activity .

How does NAT8L function in adipose tissue differ from its role in neural tissue?

NAT8L exhibits tissue-specific functions that can be investigated using distinct experimental approaches:

• Adipose tissue functions:

  • Highly expressed in adipocytes with expression induced during differentiation of mouse and human adipogenic cells

  • Accelerates lipid turnover (increased glucose incorporation into neutral lipids coupled with enhanced lipolysis)

  • Increases mitochondrial mass, number, and oxygen consumption

  • Elevates expression of brown adipocyte marker genes (PRDM16, CIDEA, PGC1α, PPARα, and UCP1)

  • Influences energy expenditure through PPARα-dependent mechanisms

• Neural tissue functions:

  • Primarily known for NAA synthesis, essential for myelination

  • Regulates dopamine uptake through TNF-alpha expression modulation

  • Contributes to neuronal energy metabolism and signaling

• Methodological approaches to distinguish these roles:

  • Tissue-specific knockout models

  • Cell-type specific transcriptomic analysis

  • Comparative metabolomics across tissues

  • Assessment of downstream effectors (PPARα in adipose tissue vs. myelin-related factors in neural tissue)

This multi-faceted role makes NAT8L an intriguing target for metabolic research spanning both neural and adipose tissue biology, with potential implications for both neurological disorders and metabolic diseases .

What are the critical parameters for immunofluorescence/immunocytochemistry using NAT8L antibodies?

For optimal immunofluorescence results with NAT8L antibodies, researchers should consider these validated protocols:

• Sample preparation:

  • Cell types with confirmed positive detection: HEK-293 cells

  • Fixation method significantly impacts epitope accessibility (4% paraformaldehyde recommended)

  • Permeabilization optimization critical for accessing intracellular epitopes

• Protocol optimization:

  • Recommended dilution range: 1:200-1:800 for IF/ICC applications

  • Primary antibody incubation: Overnight at 4°C for best signal-to-noise ratio

  • Blocking conditions: 5-10% normal serum from the species of secondary antibody origin

  • Counterstaining: Include mitochondrial markers as NAT8L shows mitochondrial localization in brown adipocytes

• Controls and validation:

  • Peptide competition control to verify specificity

  • Subcellular localization should be consistent with NAT8L's reported mitochondrial and membrane association

  • Validate patterns across multiple cell types with known NAT8L expression profiles

These methodological considerations ensure reliable detection and proper interpretation of NAT8L subcellular distribution .

For effective siRNA-mediated NAT8L knockdown in functional studies, researchers should implement these validated approaches:

• siRNA design and selection:

  • Multiple independent siRNAs should be tested to confirm specificity

  • si(NAT8L)#1 has demonstrated the most efficient protein-level knockdown (68% reduction) in published studies

  • Target selection should consider all potential NAT8L splice variants

• Experimental validation:

  • Confirm knockdown efficiency at both mRNA (qPCR) and protein (Western blot) levels

  • Expected phenotypes include selective reduction of intracellular NAA (by ~72%) and secreted NAA levels

  • Monitor cell viability as NAT8L knockdown may affect metabolic functions

• Functional readouts:

  • Metabolite analysis: Focus on NAA, acetyl-CoA, and aspartate levels

  • Compensatory mechanisms: Monitor expression of alternative acetyl-CoA producing enzymes (e.g., ATP-citrate lyase shows increased expression in NAT8L-silenced cells)

  • Tissue-specific effects: Different outcomes expected in neural vs. adipose tissues

• Controls and interpretation:

  • Include non-targeting siRNA controls

  • Rescue experiments with siRNA-resistant NAT8L constructs to confirm specificity

  • Compare with published NAT8L knockout phenotypes when available

This methodological approach has been successfully employed to demonstrate NAT8L's specific role in NAA production in various cell types .

How can NAT8L antibodies be applied in cancer biomarker research?

NAT8L antibodies offer promising applications in cancer biomarker research, with several methodological approaches:

• Tissue-based applications:

  • Immunohistochemical analysis of tumor microarrays to correlate NAT8L expression with clinical outcomes

  • Evaluation of NAT8L as a diagnostic marker in NSCLC (40-44% of adenocarcinomas show elevated expression)

  • Potential for developing companion diagnostics for therapies targeting NAA-dependent metabolic pathways

• Liquid biopsy development:

  • NAA blood levels are elevated in 46% of NSCLC patients under 55 years of age

  • Combined detection of NAT8L in circulating tumor cells and NAA in plasma may enhance diagnostic sensitivity

  • Age-stratified analysis is critical as NAA blood levels naturally increase in individuals over 56 years

• Methodological considerations:

  • Standardization of detection methods across laboratories

  • Integration with other cancer biomarkers for improved specificity

  • Longitudinal studies to assess NAT8L/NAA dynamics during disease progression and treatment response

The cancer-specific expression pattern of NAT8L makes it particularly valuable for developing highly specific cancer biomarkers with potential applications in early detection and treatment monitoring .

What metabolic pathways intersect with NAT8L function that might influence experimental design?

NAT8L intersects with several key metabolic pathways that should be considered when designing NAT8L-focused experiments:

• Acetyl-CoA metabolism:

  • NAT8L utilizes acetyl-CoA for NAA synthesis, potentially affecting acetyl-CoA availability for other processes

  • In NAT8L knockdown scenarios, ATP-citrate lyase expression increases as a compensatory mechanism to maintain acetyl-CoA pools

  • Experimental design should monitor acetyl-CoA levels and related enzymes (acetyl-CoA synthetase, citrate lyase)

• Amino acid metabolism:

  • L-aspartate serves as a direct substrate for NAT8L

  • NAT8L activity may influence aspartate availability for other cellular processes

  • Nutrient availability (especially glutamine) impacts NAA biosynthesis in NSCLC cells

• Lipid metabolism:

  • In adipocytes, NAT8L overexpression increases glucose incorporation into neutral lipids

  • NAT8L influences mitochondrial biogenesis and lipid turnover in brown adipocytes

  • PPARα-dependent pathways mediate some NAT8L effects on energy metabolism

• Dopaminergic signaling:

  • NAT8L regulates dopamine uptake via TNF-alpha expression

  • Experiments in neural systems should consider potential effects on neurotransmitter dynamics

These metabolic intersections demonstrate the importance of comprehensive metabolic profiling when studying NAT8L function, particularly in cancer and adipose tissue contexts .

What are the emerging applications of NAT8L research in metabolic diseases?

Recent discoveries about NAT8L's role in adipose tissue metabolism suggest promising new research directions:

• Brown adipose tissue activation:

  • NAT8L overexpression increases expression of brown adipocyte marker genes (UCP1, PGC1α, PPARα)

  • Enhanced mitochondrial biogenesis and oxygen consumption suggest potential for increasing energy expenditure

  • Research opportunities exist for exploring NAT8L as a target to enhance thermogenesis in metabolic disorders

• Lipid metabolism modulation:

  • NAT8L influences lipid turnover (both synthesis and lipolysis)

  • The NAT8L/NAA pathway may provide alternative sources of acetyl-CoA for lipid biosynthesis

  • Potential therapeutic relevance for dysregulated lipid metabolism in obesity and related disorders

• Methodological approaches:

  • Adipose-specific conditional knockout models

  • Pharmacological modulation of NAT8L activity

  • Metabolic phenotyping under various nutritional and environmental conditions

  • Integration with whole-body energy homeostasis studies

• Translational potential:

  • The identification that "modulating this pathway could be a valuable new approach to increase energy dissipation in (brown) adipocytes" suggests therapeutic potential

  • Biomarker development for metabolic health assessment

  • Drug discovery targeting NAT8L activation in adipose tissue

These emerging applications highlight NAT8L as a novel metabolic regulator with potential relevance to obesity, diabetes, and related metabolic disorders .