NTAN1 Antibody

What is NTAN1 and what role does it play in cellular processes?

NTAN1 functions as a tertiary destabilizing enzyme in the N-end rule pathway, a step-wise process of protein degradation. It specifically deamidates N-terminal L-Asn residues on proteins to produce N-terminal L-Asp, which are subsequently conjugated to L-Arg by ATE1. These L-Arg-conjugated proteins are then recognized by specific E3 ubiquitin ligases and targeted to the proteasome for degradation . This pathway is evolutionarily conserved and has emerged as a key regulator of various cellular processes, including apoptosis regulation .

What are the common specifications of commercially available NTAN1 antibodies?

Most commercial NTAN1 antibodies are rabbit polyclonal antibodies purified through antigen affinity methods. They typically have these specifications:

What species reactivity do NTAN1 antibodies typically demonstrate?

Most commercially available NTAN1 antibodies show reactivity against human NTAN1, with some also cross-reacting with mouse NTAN1 . When selecting an antibody for your research, it's important to verify the specific species reactivity needed for your experimental model. The polyclonal antibody described in search result demonstrates reactivity to both human and mouse NTAN1, making it suitable for comparative studies across these species.

What applications are NTAN1 antibodies validated for?

NTAN1 antibodies are validated for several standard immunological applications:

Each application requires specific optimization for your experimental system to achieve optimal signal-to-noise ratios.

How should researchers optimize NTAN1 antibody dilutions for different applications?

Optimization of antibody dilutions is crucial for obtaining specific signals while minimizing background. For NTAN1 antibodies, start with the manufacturer's recommended dilutions (IHC 1:40-1:200, ELISA 1:5000-1:10000) and perform a dilution series to determine the optimal concentration for your specific sample type and detection method. When optimizing:

• Prepare a serial dilution series (e.g., 1:20, 1:50, 1:100, 1:200 for IHC)

• Include positive controls (tissues/cells known to express NTAN1)

• Include negative controls (secondary antibody only, isotype controls)

• Evaluate both signal intensity and background at each dilution

• Select the dilution that provides maximum specific signal with minimal background

Remember that optimal dilutions may vary between different lots of the same antibody.

What controls should be included when working with NTAN1 antibodies?

Proper controls are essential for validating NTAN1 antibody specificity:

These controls collectively ensure that the observed signals are truly representative of NTAN1 expression and localization.

What are the best practices for storage and handling of NTAN1 antibodies?

To maintain antibody performance over time:

• Store antibodies at -20°C in small aliquots to minimize freeze-thaw cycles

• Always keep antibodies on ice when in use

• Avoid contamination by using clean pipette tips

• Return antibodies to appropriate storage conditions immediately after use

• Monitor shelf life (typically 12 months) and check performance regularly

• For diluted working solutions, store at 4°C and use within 1-2 weeks

• Include stabilizing proteins (e.g., BSA) in working dilutions to prevent adsorption to tubes

Proper storage and handling significantly extend antibody shelf life and ensure consistent experimental results.

How does NTAN1 function in the N-end rule pathway and what methods are used to study this?

NTAN1 plays a critical role in the N-end rule pathway, which relates protein degradation to the identity of its N-terminal residue. To study this function:

• Biochemical assays: Researchers can monitor NTAN1's deamidation activity using synthetic peptides with N-terminal asparagine and measuring conversion to aspartate using HPLC or mass spectrometry.

• Protein stability assays: Create reporter constructs with different N-terminal residues and measure their half-lives in the presence or absence of NTAN1 using cycloheximide chase experiments. In these experiments, researchers observe that proteins with N-terminal asparagine exhibit extended half-lives in NTAN1-deficient conditions .

• Ubiquitination analysis: Examine the polyubiquitination status of NTAN1 substrates using immunoprecipitation followed by Western blotting with anti-ubiquitin antibodies.

• Proteasome inhibition: Use proteasome inhibitors like MG-132 to demonstrate the dependency of substrate degradation on the proteasome pathway .

The pathway functions sequentially: NTAN1 deamidates N-terminal L-Asn residues to produce N-terminal L-Asp, which are then conjugated to L-Arg by ATE1, recognized by specific E3 ubiquitin ligases, and finally targeted to the proteasome for degradation .

What is known about NTAN1's role in apoptosis regulation?

Research has revealed a significant connection between NTAN1 and apoptosis regulation:

• NTAN1 influences the stability of apoptosis regulators, particularly the Drosophila inhibitor of apoptosis 1 (DIAP1) .

• During viral infection, degradation of NTAN1 leads to the accumulation of caspase-cleaved DIAP1, which inhibits apoptosis .

• Experimental evidence shows that:

  • Loss of NTAN1 prevents degradation of cleaved DIAP1

  • Restoration of NTAN1 expression promotes apoptosis and increases caspase activity

  • NTAN1 knockdown inhibits virus-induced apoptosis and enhances viral replication

To study this connection, researchers can:

• Use NTAN1 knockdown/overexpression approaches

• Monitor caspase activity with fluorogenic substrates

• Assess apoptosis via Annexin V/PI staining

• Measure levels of apoptotic markers by Western blot

This relationship demonstrates how the N-end rule pathway intersects with cellular apoptotic machinery, revealing NTAN1 as a potential target for manipulating cell death pathways.

How can researchers detect endogenous versus overexpressed NTAN1?

When studying both endogenous and overexpressed NTAN1, researchers should consider:

When using epitope-tagged constructs, researchers should verify that the tag doesn't interfere with NTAN1 function or localization. Western blot can distinguish between endogenous and overexpressed NTAN1 based on molecular weight differences due to the tag.

How does viral infection affect NTAN1 stability and function?

Viral infection has been shown to significantly impact NTAN1 regulation, particularly in the context of a picorna-like virus model :

• Viral infection induces gradual decrease of NTAN1 protein levels while paradoxically upregulating NTAN1 mRNA expression .

• The mechanism involves post-transcriptional regulation, as both endogenous and exogenously expressed NTAN1 are degraded during viral infection .

• The degradation is proteasome-dependent but polyubiquitylation-independent, suggesting an unconventional degradation pathway .

• Functional consequences include:

  • Accumulation of caspase-cleaved DIAP1

  • Inhibition of apoptosis

  • Enhanced viral replication

This virus-induced suppression of the N-end rule pathway represents a novel mechanism for viral evasion of host cell apoptosis. Researchers can monitor this phenomenon using:

• Time-course experiments measuring NTAN1 protein levels after infection

• Proteasome inhibitors to block degradation

• mRNA vs. protein expression analysis

• Viral titer measurements with NTAN1 restoration

What role do specific lysine residues play in NTAN1 regulation?

Research has identified critical lysine residues that regulate NTAN1 stability:

Interestingly, while NTAN1 wild-type can be polyubiquitylated, this polyubiquitylation is not affected by viral infection, supporting the existence of parallel degradation pathways . Researchers studying NTAN1 regulation should consider:

• Using site-directed mutagenesis to generate lysine mutants

• Comparing stability of wild-type vs. mutant proteins

• Assessing ubiquitylation status under various conditions

• Investigating proteasome-dependent but ubiquitin-independent degradation mechanisms

What experimental approaches can be used to study NTAN1's interaction with substrates?

To investigate NTAN1's interaction with its substrates, researchers can employ multiple complementary approaches:

• Direct binding assays:

  • Co-immunoprecipitation using NTAN1 antibodies to pull down interacting partners

  • GST pull-down assays with recombinant NTAN1

  • Yeast two-hybrid screening to identify novel interactors

• Functional assays:

  • In vitro deamidation assays with purified NTAN1 and substrate proteins

  • Cyclohexamide chase experiments to measure substrate stability

  • Comparative proteomics between wild-type and NTAN1-deficient cells

• Structural studies:

  • X-ray crystallography or cryo-EM to determine NTAN1-substrate complex structures

  • Hydrogen-deuterium exchange mass spectrometry to map interaction interfaces

  • Computational docking to predict binding modes

• Live cell approaches:

  • FRET/BRET to monitor real-time interactions

  • BiFC (Bimolecular Fluorescence Complementation) to visualize interactions in cells

  • Proximity ligation assays to detect endogenous protein interactions

These methodologies collectively provide a comprehensive understanding of how NTAN1 recognizes and processes its substrates within the N-end rule pathway.

What are common issues when using NTAN1 antibodies and how can they be resolved?

Researchers may encounter several challenges when working with NTAN1 antibodies:

For optimal results, always validate new antibody lots against previous standards and include appropriate positive and negative controls in every experiment.

How can researchers verify NTAN1 antibody specificity?

Verifying antibody specificity is crucial for reliable results. For NTAN1 antibodies, employ these approaches:

• Genetic validation:

  • Use NTAN1 knockout/knockdown samples as negative controls

  • Compare signal in NTAN1-overexpressing vs. control cells

• Biochemical validation:

  • Peptide competition assays with the immunizing peptide/protein

  • Test multiple antibodies targeting different NTAN1 epitopes

  • Immunoprecipitation followed by mass spectrometry

• Application-specific validation:

  • For Western blot: Verify band corresponds to expected molecular weight

  • For IHC/IF: Compare with known expression patterns

  • For ELISA: Establish standard curves with recombinant NTAN1

• Cross-technique validation:

  • Confirm findings across multiple techniques (e.g., WB, IHC, and IF)

  • Correlate protein detection with mRNA expression data

Thorough validation ensures that experimental observations truly reflect NTAN1 biology rather than antibody artifacts.