NAA15 Antibody, Biotin conjugated

What is NAA15 and why is it significant in research applications?

NAA15 (N-alpha-acetyltransferase 15) functions as the auxiliary subunit of the NatA complex, a major N-terminal acetyltransferase that modifies approximately 40-50% of mammalian proteins. NAA15 is also known by several alternative names including gastric cancer antigen Ga19 (GA19), NMDA receptor-regulated protein 1 (NARG1), and Tbdn100 . The protein positions the catalytic subunits (primarily NAA10) in proximity to nascent polypeptides emerging from the ribosome exit tunnel, modulating substrate specificity and facilitating N-terminal acetylation .

Research significance:

• NAA15 haploinsufficiency has been linked to congenital heart defects and neurodevelopmental disorders

• NAA15 knockdown has been shown to enhance C2C12 myoblast fusion

• NAA15 forms complexes not only with NAA10 but also with NAA11, NAA12, and other proteins

These diverse functions make NAA15 antibodies valuable tools for investigating protein acetylation mechanisms, developmental biology, and disease pathogenesis.

What are the technical advantages of biotin-conjugated NAA15 antibodies over unconjugated versions?

Biotin-conjugated NAA15 antibodies offer several methodological advantages:

• Enhanced signal amplification: The biotin-avidin/streptavidin system provides one of the strongest non-covalent biological interactions (Kd ≈ 10^-15 M), allowing for significant signal enhancement in detection systems.

• Compatibility with multiple detection systems: The biotin tag enables versatile detection methods including:

  • Streptavidin-HRP for chemiluminescent detection

  • Streptavidin-fluorophore conjugates for fluorescent detection

  • Streptavidin-gold for electron microscopy applications

• Reduced background in tissue samples: The biotin-conjugated format often produces cleaner results in immunohistochemistry by avoiding direct enzyme conjugation to the primary antibody.

• Stable conjugation chemistry: The biotin moiety maintains stability during storage and experimental conditions, providing reliable detection capabilities .

• Multiplexing capability: The biotin tag facilitates incorporation into multi-antibody detection systems without interfering with antigen recognition sites.

What are optimal storage conditions for maintaining NAA15 antibody activity?

For maximum retention of immunoreactivity, NAA15 antibodies should be stored according to these guidelines:

• Short-term storage (up to 1 week): Store at 4°C in their original buffer containing preservatives (typically PBS with 0.02% sodium azide) .

• Long-term storage: Aliquot and store at -20°C. Avoid repeated freeze-thaw cycles as these significantly diminish antibody activity .

• Working solution preparation: Dilutions should be prepared fresh before use and generally should not be stored for future applications.

• Buffer composition: The typical storage buffer contains PBS with 0.02-0.05% sodium azide and may include 50% glycerol for cryoprotection .

Proper storage significantly affects experimental reproducibility, particularly for quantitative applications such as western blotting or ELISA where signal intensity directly correlates with functional antibody concentration.

What dilutions are recommended for biotin-conjugated NAA15 antibodies in various applications?

Based on the technical specifications, the following dilution ranges are recommended for biotin-conjugated NAA15 antibodies:

These dilutions serve as starting points and should be optimized for each experimental system. Researchers should perform dilution series to determine the optimal antibody concentration that produces specific signal with minimal background.

How does epitope selection affect experimental outcomes when using NAA15 antibodies?

The epitope recognized by the NAA15 antibody significantly impacts experimental results:

• N-terminal vs. internal epitopes: The biotin-conjugated NAA15 antibody specified in the search results targets N-terminal epitopes , which may be less accessible when NAA15 is incorporated into the NatA complex with NAA10 or NAA12 . This potential masking effect should be considered when studying protein interactions.

• Isoform detection: NAA15 has multiple reported isoforms , and epitope selection determines which variants can be detected. Antibodies targeting amino acids 225-269 of human NAA15 will detect the canonical 866 amino acid form but may miss shorter isoforms.

• Cross-species reactivity: The homology between human, mouse, and rat NAA15 affects epitope conservation. When working with animal models, researchers should verify reactivity through preliminary experiments or western blotting of tissue lysates .

• Post-translational modifications: Epitopes containing modification sites may affect antibody recognition depending on the protein's modification state. For accurate interpretation, researchers should consider potential phosphorylation, acetylation, or other modifications near the epitope region.

What methodological approaches can be used to validate NAA15 antibody specificity?

Rigorous validation is essential for reliable research outcomes. The following approaches are recommended:

• Peptide competition assays: Pre-incubate the NAA15 antibody with blocking peptides containing the epitope sequence. This neutralizes specific binding, allowing differentiation between specific and non-specific signals. As noted in search result , "Specific binding will be absent from the western blot or IHC performed with the neutralized antibody."

• Genetic approaches:

  • Use NAA15 knockdown (siRNA) samples as negative controls

  • Compare wild-type samples with NAA15 knockout or haploinsufficient models

  • Include NAA15-overexpressing samples as positive controls

• Cross-validation with multiple antibodies: Use antibodies targeting different NAA15 epitopes to confirm consistent detection patterns.

• Immunoprecipitation followed by mass spectrometry: Confirm the identity of immunoprecipitated proteins to verify antibody specificity.

• Western blot analysis: Verify detection of the expected ~101 kDa band for full-length NAA15 , along with any known isoforms. Example western blot data from search result demonstrates detection of NAA15 in:

  • A549 whole cell lysate (20μg)

  • L02 whole cell lysate (20μg)

  • Mouse testis tissue lysate (40μg)

  • Rat testis tissue lysate (40μg)

How can NAA15 antibodies be utilized in studying congenital heart defects and neurodevelopmental disorders?

NAA15 has been implicated in both congenital heart defects (CHD) and neurodevelopmental disorders. Methodological approaches using NAA15 antibodies include:

• Tissue expression profiling:

  • Immunohistochemistry of cardiac tissue sections at different developmental stages

  • Comparison of NAA15 expression in normal versus CHD-affected tissues

  • Brain region-specific expression analysis in neurodevelopmental contexts

• Mechanistic studies:

  • Co-immunoprecipitation using NAA15 antibodies to identify protein interaction partners in cardiac and neural tissues

  • Chromatin immunoprecipitation (ChIP) to investigate transcriptional regulation if NAA15 shows nuclear localization

  • Protein acetylation profiling to correlate with NAA15 expression levels

• iPSC-based disease modeling:

  • Detection of NAA15 in patient-derived induced pluripotent stem cells (iPSCs)

  • Monitoring NAA15 expression during cardiac or neuronal differentiation

  • Comparison between wild-type and NAA15 variant-containing iPSCs for protein interaction differences

• Protein complex analysis:

  • Investigation of NatA complex integrity (NAA15-NAA10 interaction) in disease models

  • Quantification of complex components (NAA15, NAA10, HYPK, NAA50) in patient samples

  • Analysis of N-terminal acetylation patterns in proteins associated with cardiac development

Research has shown that NAA15 haploinsufficiency leads to variable levels of intellectual disability, delayed speech and motor milestones, autism spectrum disorder, and in some cases, congenital cardiac anomalies .

What technical considerations apply when using biotin-conjugated NAA15 antibodies in multiplex immunoassays?

When incorporating biotin-conjugated NAA15 antibodies into multiplex detection systems:

• Endogenous biotin interference:

  • Tissues with high endogenous biotin (brain, liver, kidney) may produce background signal

  • Pretreatment with avidin/biotin blocking systems is essential for these samples

  • Heat-induced epitope retrieval may alter endogenous biotin accessibility

• Panel design considerations:

  • Avoid other biotin-conjugated antibodies in the same panel unless using sequential detection methods

  • Ensure secondary detection reagents (streptavidin conjugates) don't cross-react with other primary antibodies

  • Consider steric hindrance when NAA15 is part of larger protein complexes

• Signal amplification optimization:

  • Titrate streptavidin detection reagents to prevent signal saturation

  • When using tyramide signal amplification (TSA), carefully control reaction times

  • Use appropriate negative controls to establish baseline signal

• Cross-reactivity assessment:

  • Validate absence of cross-reactivity with other N-acetyltransferase complex components

  • Confirm specificity in the presence of related proteins (NAA10, NAA11, NAA12)

• Detection system compatibility:

  • Ensure imaging systems can accommodate detection wavelengths for chosen streptavidin conjugates

  • Consider spectral overlap when designing multiplex panels

  • Validate signal separation through single-stain controls

How can NAA15 antibodies be used to study the relationship between N-terminal acetylation and protein function?

NAA15 antibodies provide valuable tools for investigating acetylation mechanisms:

• Co-immunoprecipitation approaches:

  • Use NAA15 antibodies to pull down NatA complexes

  • Identify substrate proteins via mass spectrometry

  • Compare acetylated protein profiles between wild-type and NAA15-deficient samples

• Acetylation site mapping:

  • Combine NAA15 immunoprecipitation with acetylome analysis

  • Correlate NAA15 expression levels with changes in N-terminal acetylation patterns

  • Identify NAA15-dependent acetylation sites through differential analysis

• Subcellular localization studies:

  • Use NAA15 antibodies to track localization to ribosomes

  • Investigate co-localization with emerging polypeptide chains

  • Analyze spatial distribution of acetylation machinery in various cell types

• Functional impact assessment:

  • Compare protein stability in NAA15 knockdown versus control cells

  • Investigate protein-protein interaction differences based on acetylation status

  • Analyze downstream signaling pathway alterations following NAA15 modulation

• Developmental regulation:

  • Track NAA15 expression during differentiation processes

  • Correlate with changes in substrate protein acetylation patterns

  • Investigate temporal regulation of NatA complex formation

Research has shown that mass spectrometry analyses reveal approximately 80% of identified iPSC NatA targeted proteins displayed partial or complete N-terminal acetylation, with N-terminal acetylation levels of 32 and 9 NatA-specific targeted proteins reduced in null and haploinsufficient NAA15 cells, respectively .

How do rabbit polyclonal NAA15 antibodies compare to antibodies from other host species?

While the search results primarily discuss rabbit polyclonal NAA15 antibodies , comparing antibody sources provides important methodological context:

The rabbit polyclonal format provides advantages for detecting low-abundance proteins and offers flexibility across applications, but requires careful validation for specificity.

What troubleshooting approaches are recommended when NAA15 antibody experiments produce unexpected results?

When facing technical challenges with NAA15 antibody experiments:

• No signal detected:

  • Verify NAA15 expression in the sample type (NAA15 is notably expressed at high levels in testis and ocular endothelial cells)

  • Increase antibody concentration or extend incubation time

  • Check detection system functionality with positive controls

  • Consider epitope accessibility issues if samples are fixed or cross-linked

  • Try alternative epitope retrieval methods

• Multiple unexpected bands:

  • Determine if bands represent known isoforms (NAA15 has multiple reported isoforms)

  • Test specificity with blocking peptides

  • Optimize sample preparation to reduce protein degradation

  • Increase washing stringency to reduce non-specific binding

  • Compare with NAA15 knockout/knockdown samples if available

• High background:

  • Increase blocking concentration or time

  • For biotin-conjugated antibodies, use avidin/biotin blocking in tissues with high endogenous biotin

  • Reduce primary antibody concentration

  • Extend washing steps duration and number

  • Consider alternative detection methods

• Cross-reactivity concerns:

  • Validate with additional NAA15 antibodies targeting different epitopes

  • Confirm specificity using genetic models (NAA15 knockdown/knockout)

  • Perform pre-absorption controls with recombinant NAA15 protein

How can NAA15 antibodies be utilized to investigate differences between NAA15 and its paralogs?

NAA15 has several paralogs and interaction partners that can be studied using antibody-based approaches:

• Differential expression analysis:

  • Compare expression patterns of NAA15 versus NAA11 (paralog) in various tissues

  • Investigate co-expression with the recently discovered NAA12 (NAA10-like paralog)

  • Analyze relative levels of NAA15 and its binding partners (NAA10, NAA50, HYPK) in different cell types

• Complex formation studies:

  • Use co-immunoprecipitation with NAA15 antibodies to pull down different NatA complexes

  • Compare complex composition between tissues/developmental stages

  • Identify tissue-specific interaction partners

• Functional complementation analysis:

  • Investigate NAA15 expression in NAA12 knockout models to assess compensation

  • Compare acetylation activity in immunoprecipitated complexes containing different paralogs

  • Study localization patterns of different NAA15-containing complexes

• Developmental regulation:

  • Track expression timing of NAA15 versus paralogs during embryonic development

  • Correlate with phenotypic outcomes in genetic models

  • Analyze tissue-specific expression patterns

• Cross-species comparative studies:

  • Use NAA15 antibodies that cross-react with multiple species to compare conservation of expression patterns

  • Analyze differences in complex formation across evolutionary distance

  • Correlate with functional conservation of N-terminal acetylation machinery

The discovery of NAA12 adds complexity to our understanding of N-terminal acetylation machinery, as mice deficient for NAA10 show no globally apparent in vivo amino-terminal acetylation impairment, likely due to compensation by NAA12 .

What methodological approaches can be used to study NAA15's role in myoblast fusion using antibody-based techniques?

Building on the finding that NAA15 knockdown enhances C2C12 myoblast fusion , researchers can implement these antibody-based protocols:

• Temporal expression profiling:

  • Track NAA15 protein levels during myoblast differentiation using western blotting

  • Correlate expression changes with fusion index measurements

  • Compare with markers of myogenic differentiation (MyoG, MCK)

• Subcellular localization analysis:

  • Use immunofluorescence with NAA15 antibodies to track protein distribution before and during fusion

  • Co-stain with markers of the secretory pathway to investigate potential involvement in fusion-related trafficking

  • Analyze relocalization during fusion events

• Protein complex immunoprecipitation:

  • Use NAA15 antibodies to immunoprecipitate protein complexes at different stages of myoblast differentiation

  • Identify fusion-specific interaction partners through mass spectrometry

  • Validate interactions using reciprocal co-immunoprecipitation

• Chromatin immunoprecipitation (ChIP):

  • If NAA15 shows nuclear localization, investigate potential transcriptional regulatory roles

  • Target fusion-related gene promoters to assess NAA15 occupancy

  • Correlate with expression changes in fusion-related genes

• Rescue experiments:

  • After NAA15 knockdown, reintroduce wild-type or mutant NAA15

  • Use antibodies to confirm expression levels

  • Correlate with restoration or enhancement of fusion phenotypes

The C2C12-MCK:GFP mouse myoblast cell line provides an excellent model system for these studies, as the GFP reporter enables monitoring of differentiation in parallel with NAA15 detection .

How can researchers optimize immunoprecipitation protocols using biotin-conjugated NAA15 antibodies?

For effective immunoprecipitation with biotin-conjugated NAA15 antibodies:

• Capture strategy optimization:

  • Direct approach: Use streptavidin-coated magnetic beads to capture biotin-conjugated NAA15 antibodies bound to target protein

  • Indirect approach: Pre-bind biotin-conjugated antibodies to streptavidin beads before sample addition

  • Compare efficiency of both methods in your experimental system

• Buffer composition considerations:

  • For NatA complex isolation: Use buffers that preserve protein-protein interactions (150-300mM NaCl, 0.1-0.5% NP-40)

  • For stringent conditions: Increase salt concentration (up to 500mM) and detergent percentage

  • Include protease inhibitors to prevent complex degradation

  • Consider adding N-acetylation inhibitors to preserve modification states

• Sequential elution strategies:

  • For complex analysis: Use mild elution conditions to maintain interactions

  • For interactome studies: Use biotin for competitive elution to preserve antibody-antigen binding

  • For subsequent mass spectrometry: Consider on-bead digestion to minimize sample loss

• Validation approaches:

  • Confirm successful precipitation by western blotting for NAA15

  • Verify co-precipitation of known interaction partners (NAA10, NAA50, HYPK)

  • Include appropriate negative controls (non-specific IgG or unrelated biotin-conjugated antibodies)

• Cross-linking options:

  • Consider membrane-permeable crosslinkers for capturing transient interactions

  • Optimize crosslinking time to balance capturing complexes versus creating non-specific aggregates

  • Include appropriate controls for crosslinking efficiency

What experimental design considerations are important when using NAA15 antibodies in iPSC-based disease modeling?

When incorporating NAA15 antibodies into iPSC disease modeling experiments:

• Temporal analysis requirements:

  • Monitor NAA15 expression throughout differentiation processes

  • Compare expression timing between wild-type and disease-relevant NAA15 variant lines

  • Correlate with developmental markers to identify critical expression windows

• Differentiation protocol influences:

  • NAA15 has been implicated in both cardiac and neural development

  • Different differentiation protocols may affect NAA15 expression patterns

  • Compare directed versus spontaneous differentiation outcomes

• Validation in multiple iPSC lines:

  • Use multiple patient-derived and control lines to account for genetic background effects

  • Include isogenic controls (CRISPR-corrected lines) when possible

  • Compare with embryonic stem cell lines as reference standards

• Technical controls:

  • Include NAA15 haploinsufficient or knockout iPSC lines as controls for antibody specificity

  • Validate antibody performance in pluripotent versus differentiated states

  • Monitor potential changes in epitope accessibility during differentiation

• Functional readouts:

  • Correlate NAA15 detection with measurements of N-terminal acetylation status

  • Assess impact on interacting proteins (NAA10, HYPK)

  • Connect to phenotypic outcomes (differentiation efficiency, structural abnormalities)

Research has demonstrated that NAA15 iPSCs can differentiate into cardiomyocytes, unlike NAA15-null iPSCs, highlighting the importance of careful genetic model selection for these studies .