NAT10 Antibody, HRP conjugated

What is NAT10 and what are its primary cellular functions?

NAT10 (N-Acetyltransferase 10) functions as an RNA cytidine acetyltransferase that catalyzes the formation of ac4C RNA modifications. This enzyme plays critical roles in multiple cellular processes including T cell development and homeostasis . NAT10 is the sole known enzyme responsible for ac4C modification, making it particularly significant in RNA metabolism research . Recent studies have demonstrated that NAT10 maintains T cell balance through the NAT10-ac4C-Bag3 axis, which regulates apoptosis and cell survival pathways . Additionally, NAT10 has been implicated in cancer development, with upregulation observed in hepatocellular carcinoma where it enhances mutant p53 stability .

What are the optimal storage and handling practices for NAT10 Antibody, HRP conjugated?

For maximum stability and activity retention, NAT10 Antibody, HRP conjugated should be stored at -20°C in aliquots to avoid repeated freeze-thaw cycles that can significantly reduce activity . The product is supplied in a buffer containing 0.01M PBS, pH 7.4, with 0.03% Proclin-300 and 50% glycerol for stability . When handling the antibody:

• Protect from light exposure to prevent photobleaching of the HRP conjugate

• Avoid repeated freeze-thaw cycles by preparing single-use aliquots

• Thaw aliquots at room temperature immediately before use

• Centrifuge briefly before opening to collect content at the bottom of the tube

• Return unused portion to -20°C immediately after use

Each freeze-thaw cycle can reduce antibody activity by approximately 10-15%, so proper aliquoting is essential for maintaining consistent experimental results.

What methodological considerations should be addressed when using NAT10 Antibody, HRP conjugated for ELISA applications?

The NAT10 Antibody, HRP conjugated has been specifically tested for ELISA applications . When employing this antibody in ELISA protocols, researchers should consider:

• Optimization of antibody dilution: While the manufacturer indicates that optimal dilutions should be determined by the end user , a recommended starting range is typically 1:500 to 1:5000 for HRP-conjugated antibodies in ELISA.

• Blocking protocol optimization: To reduce background signal, use a blocking buffer containing 1-5% BSA or normal serum from the same species as the secondary antibody.

• Substrate selection: Choose an appropriate substrate based on required sensitivity:

  • TMB (3,3',5,5'-Tetramethylbenzidine) for colorimetric detection

  • ECL substrates for higher sensitivity via chemiluminescence

• Incubation conditions: Optimize temperature (typically 4°C overnight or room temperature for 1-2 hours) and duration to maximize signal-to-noise ratio.

• Washing steps: Include sufficient washing steps (typically 3-5 washes) with PBS-T (0.05% Tween-20) to remove unbound antibody and reduce background.

• Positive and negative controls: Include well-characterized samples with known NAT10 expression levels to validate assay performance.

How can researchers validate NAT10 antibody specificity in their experimental systems?

Validating antibody specificity is crucial for generating reliable scientific data. For NAT10 Antibody, HRP conjugated, recommended validation approaches include:

• Knockdown/knockout controls: Compare antibody signal in samples with NAT10 knockdown (using shRNA as described in search results ) versus control samples. A specific antibody will show significantly reduced signal in NAT10-depleted samples.

• Comparison with alternative antibodies: Test multiple antibodies targeting different epitopes of NAT10 to confirm consistent detection patterns.

• Blocking peptide competition: Pre-incubate the antibody with the immunogen peptide (recombinant human RNA cytidine acetyltransferase protein, amino acids 909-1025 ) before application to samples. A specific signal should be significantly reduced.

• Western blot analysis: Confirm the antibody detects a band of appropriate molecular weight (~115 kDa for human NAT10).

• Mass spectrometry validation: For advanced validation, immunoprecipitate the target and confirm identity via mass spectrometry.

These validation steps are particularly important considering NAT10's critical role in fundamental cellular processes where research findings have significant implications.

How can NAT10 Antibody, HRP conjugated be optimized for detecting NAT10 in cancer research?

NAT10 has demonstrated significant upregulation in hepatocellular carcinoma, with 73.7% of tumor samples showing increased NAT10 protein levels compared to matched non-cancerous tissues . When investigating NAT10 in cancer contexts:

What are the key considerations when investigating NAT10's role in T cell development using the HRP-conjugated antibody?

Recent research has revealed NAT10's critical role in T cell development and homeostasis . When designing experiments to study this relationship:

• Temporal expression analysis: NAT10 expression increases progressively during T cell activation, with significant changes observed from 0-96 hours post-activation . Experimental designs should:

  • Include multiple time points for capturing the dynamic expression pattern

  • Compare activation states (naïve versus activated T cells)

  • Consider both protein and RNA expression analysis

• Functional correlation studies: Link NAT10 expression/activity to specific T cell functions:

  • Proliferation (correlate with Ki67 expression)

  • Apoptosis (measure Annexin V expression)

  • Migration capacity (assess CXCR3 expression)

  • Cytokine production (monitor IFN-γ and granzyme B)

• RNA modification analysis: As NAT10 catalyzes the only known RNA acetylation (ac4C), consider:

  • Parallel analysis of ac4C levels and targets in T cells

  • RNA stability assays to assess functional impact of NAT10-mediated modifications

• Pathway integration: Data suggest NAT10 maintains T cell homeostasis through regulating anti-apoptotic gene Bag3 , so experimental designs should include assessment of this pathway.

How can researchers use NAT10 Antibody, HRP conjugated to study its role in apoptosis regulation?

NAT10 plays a significant role in apoptosis regulation as evidenced by several studies. When designing experiments to investigate this function:

• Cell line selection: Consider using AML cell lines (U937, MV4-11) where NAT10 inhibition has been shown to promote apoptosis , or T cells where NAT10 deficiency increases apoptosis through Bag3 downregulation .

• Experimental approach:

  • Compare apoptosis markers (Annexin V, caspase activation) between NAT10-depleted and control cells

  • Assess expression of apoptosis regulators such as Bax/Bcl-2 axis proteins, which are activated when targeting NAT10

  • Monitor ER stress markers (GRP78, cleaved caspase-12) that increase upon NAT10 inhibition

• Mechanistic investigation:

  • Examine the Unfolded Protein Response (UPR) pathway components (IRE1, CHOP, PERK) that show increased expression when NAT10 is targeted

  • Assess the stability and expression of anti-apoptotic gene Bag3, which is regulated by NAT10

• Manipulation strategies:

  • Use NAT10 shRNA knockdown (validated approaches described in )

  • Consider pharmacological inhibition with Remodelin hydrobromide

  • Implement rescue experiments by overexpressing key downstream targets like Bag3

A comprehensive experimental approach should incorporate both NAT10 expression analysis and functional assessment of apoptotic pathways to establish causative relationships.

What experimental designs are optimal for studying NAT10's interaction with p53 using the HRP-conjugated antibody?

NAT10 has been shown to interact with and stabilize mutant p53 in hepatocellular carcinoma, affecting cancer progression . To investigate this interaction:

• Co-localization studies:

  • Immunofluorescence analysis shows NAT10 partially colocalizes with p53 in nucleoli

  • Use cell fractionation to confirm nuclear localization of NAT10 before performing interaction studies

• Protein-protein interaction analysis:

  • Co-immunoprecipitation approaches have confirmed NAT10 binding to mutant p53 in HCC cell line Huh7

  • When designing Co-IP experiments, consider nuclear extraction protocols to enrich for the relevant cellular fraction

• Functional stability studies:

  • NAT10 depletion decreases mutant p53 levels and increases its ubiquitination

  • Design ubiquitination assays to assess how NAT10 affects p53 protein stability

  • Include Mdm2 analysis, as NAT10 may counteract Mdm2-mediated degradation of mutant p53

• Cancer context considerations:

  • Since 84.2% of HCC tumor samples showed p53 upregulation (suggesting mutations) , use appropriate cell models carrying p53 mutations

  • Consider correlation analysis between NAT10 and p53 levels in patient samples

These experimental approaches should be integrated to establish both physical interaction and functional consequences of the NAT10-p53 relationship.

What are common technical challenges when using NAT10 Antibody, HRP conjugated, and how can they be addressed?

When working with NAT10 Antibody, HRP conjugated, researchers may encounter several technical challenges:

• High background signal:

  • Cause: Insufficient blocking or excessive antibody concentration

  • Solution: Optimize blocking conditions using 3-5% BSA in TBS/PBS; titrate antibody concentration starting with higher dilutions (1:2000)

• Weak signal strength:

  • Cause: Insufficient antigen, over-diluted antibody, or degraded HRP activity

  • Solution: Confirm NAT10 expression in positive control samples; store antibody properly to preserve HRP activity; optimize antigen retrieval for tissue samples

• Non-specific bands in Western blot:

  • Cause: Cross-reactivity with related proteins

  • Solution: Increase washing steps; use more stringent blocking conditions; validate with NAT10 knockdown controls

• Variable results between experiments:

  • Cause: Inconsistent handling or freeze-thaw cycles

  • Solution: Prepare single-use aliquots; standardize protocols; include consistent positive controls

• Poor detection in specific sample types:

  • Cause: Sample-specific interfering factors or low expression

  • Solution: Consider enrichment strategies (e.g., nuclear fractionation for NAT10); test alternative detection substrates with higher sensitivity

Each troubleshooting approach should be systematically documented to build reliable protocols for NAT10 detection across different experimental contexts.

Expression Profiles of NAT10 Across Different Cell Lines and Tissues

Functional Consequences of NAT10 Manipulation in Different Cellular Contexts