NAA16 Antibody

What is NAA16 and what is its biological significance?

NAA16 (N-Alpha-Acetyltransferase 16, NatA Auxiliary Subunit) is a protein-coding gene that functions as an auxiliary subunit of the N-terminal acetyltransferase A (NatA) complex. This complex is responsible for N-terminal acetylation of proteins, a major post-translational modification affecting approximately 50% of all mammalian proteins . The NAA16 protein enables ribosome binding activity and is involved in protein stabilization and negative regulation of apoptotic processes . Structurally, NAA16 is located in the cytosol and contains domains that facilitate its interaction with other components of the NatA complex .

NAA16 shares significant homology with NAA15, which is considered its important paralog . Both proteins can serve as auxiliary subunits of the NatA complex, though they may have distinct expression patterns or functional specificities depending on the cellular context. Understanding NAA16's function is particularly important for researchers studying protein acetylation mechanisms, translational regulation, and protein stability pathways in various biological systems.

What are the typical applications for NAA16 antibodies in molecular biology research?

NAA16 antibodies serve as valuable tools in multiple research applications aimed at understanding NAA16's expression, localization, interactions, and functions. The primary applications include:

• Western Blotting (WB): NAA16 antibodies enable detection and semi-quantification of NAA16 protein expression in cell or tissue lysates. This application is particularly useful for studying NAA16 expression levels under different conditions or comparing expression across different tissues or cell types .

• Immunohistochemistry (IHC): This technique allows visualization of NAA16 localization in tissue sections, providing insights into its expression patterns in different cell types within complex tissues .

• Immunocytochemistry/Immunofluorescence (ICC-IF): NAA16 antibodies can be used to examine subcellular localization of NAA16, confirming its cytosolic distribution and potentially revealing dynamic changes in localization under different cellular conditions .

• Co-immunoprecipitation (Co-IP): Researchers can use NAA16 antibodies to isolate NAA16 and its interacting partners, helping to elucidate the composition of NAA16-containing complexes and identify novel protein interactions.

• Chromatin Immunoprecipitation (ChIP): Though less common for NAA16, this application can be relevant if investigating potential chromatin associations of NAA16 or NatA complex components.

Each application requires specific optimization of antibody concentration, incubation conditions, and detection methods to ensure specific and reliable results.

What is the difference between polyclonal and monoclonal NAA16 antibodies?

The choice between polyclonal and monoclonal NAA16 antibodies depends on the specific research requirements:

Polyclonal NAA16 Antibodies:

• Produced by immunizing animals (commonly rabbits) with NAA16 protein or peptide immunogens .

• Contain a heterogeneous mixture of antibodies that recognize multiple epitopes on the NAA16 protein.

• Advantages include higher sensitivity due to binding multiple epitopes, greater tolerance to minor protein denaturation, and typically lower cost.

• Examples include rabbit polyclonal antibodies like those offered by Atlas Antibodies (HPA039761) and Assay Genie (PACO10733) .

• Most suitable for detection applications where signal amplification is important, such as Western blotting of low-abundance proteins or IHC of fixed tissues.

Monoclonal NAA16 Antibodies:

• Produced from single B-cell clones, resulting in antibodies with identical specificity targeting a single epitope.

• Advantages include exceptional specificity, reduced batch-to-batch variation, and consistent performance across experiments.

• Most appropriate for applications requiring high reproducibility, such as quantitative assays or when distinguishing between closely related proteins (e.g., NAA16 versus its paralog NAA15).

When selecting between these antibody types, researchers should consider factors such as the experimental application, required specificity, target abundance, and whether cross-reactivity with related proteins (like NAA15) might be problematic. Validation using appropriate controls is essential regardless of antibody type.

How can I validate the specificity of my NAA16 antibody?

Validating NAA16 antibody specificity is crucial for ensuring reliable experimental results. A comprehensive validation strategy includes:

• Knockdown/Knockout Controls: Perform siRNA-mediated knockdown of NAA16 (as described in the NatA studies) or use CRISPR-Cas9 to generate knockout cells . A specific antibody should show significantly reduced or absent signal in these samples compared to controls.

• Overexpression Controls: Express tagged NAA16 in cells and verify co-detection with both the NAA16 antibody and an antibody against the tag. This approach confirms that the antibody recognizes the intended target.

• Peptide Competition Assay: Pre-incubate the NAA16 antibody with the immunizing peptide before application. A specific antibody will show diminished signal when blocked with its target peptide.

• Multiple Antibody Validation: Use antibodies from different sources or those targeting different epitopes of NAA16. Consistent results across different antibodies increase confidence in specificity.

• Cross-Reactivity Assessment: Test the antibody on samples expressing NAA15 (the paralog of NAA16) to ensure it doesn't cross-react. This is particularly important given the sequence similarity between these proteins.

• Western Blot Band Pattern: Verify that the antibody detects a protein of the expected molecular weight (~50 kDa for NAA16) . Multiple unexpected bands may indicate cross-reactivity.

• Tissue/Cell Expression Pattern: Compare detected expression patterns with known NAA16 mRNA expression data from databases to confirm biological plausibility.

When documenting validation, researchers should record detailed experimental conditions and include all controls in publications to facilitate reproducibility by others in the field.

What are the best practices for optimizing NAA16 antibody concentration in Western blot experiments?

Optimizing NAA16 antibody concentration for Western blotting requires a systematic approach:

• Initial Titration Experiment:

  • Prepare a series of dilutions of your NAA16 antibody (e.g., 1:500, 1:1000, 1:2000, 1:5000, 1:10000).

  • Run identical amounts of protein lysate containing NAA16 on multiple lanes of a gel.

  • Transfer to membrane and cut into strips corresponding to each lane.

  • Probe each strip with a different antibody dilution.

  • Determine the dilution that provides the best signal-to-noise ratio, not necessarily the strongest signal.

• Sample Preparation Considerations:

  • Use freshly prepared cell lysates whenever possible.

  • Include protease inhibitors to prevent degradation of NAA16.

  • For NAA16, consider using RIPA buffer for extraction as it effectively solubilizes cytosolic proteins.

• Blocking Optimization:

  • Test different blocking agents (BSA vs. non-fat dry milk) as some antibodies perform better with specific blockers.

  • Typically, 5% blocking solution in TBST or PBST is effective for NAA16 antibodies.

• Incubation Parameters:

  • Compare overnight incubation at 4°C versus shorter incubations at room temperature.

  • For NAA16 polyclonal antibodies like those from Atlas Antibodies or Assay Genie, overnight incubation at 4°C often yields optimal results .

• Signal Development Optimization:

  • For chemiluminescent detection, adjust exposure time to avoid saturation.

  • Consider enhanced chemiluminescence (ECL) systems for low-abundance detection.

• Validation Controls:

  • Always include positive controls (cell lines known to express NAA16).

  • Consider running NAA15 as a comparison to ensure specificity.

  • If possible, include NAA16 knockdown samples as negative controls.

A methodical optimization process saves reagents and time in the long run while ensuring reproducible and reliable detection of NAA16 protein in your experimental system.

How should you store and handle NAA16 antibodies to maintain optimal activity?

Proper storage and handling of NAA16 antibodies are essential for maintaining their performance over time:

• Storage Temperature:

  • Store NAA16 antibodies at -20°C for long-term storage, as indicated for commercial antibodies like the Assay Genie PACO10733 .

  • For antibodies in use, aliquot and store at 4°C for up to 2 weeks to minimize freeze-thaw cycles.

• Buffer Composition:

  • NAA16 antibodies are typically stored in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 .

  • The glycerol prevents freezing at -20°C and reduces damage from ice crystal formation.

  • Sodium azide prevents microbial contamination but is incompatible with HRP-based detection systems, so avoid direct contact.

• Aliquoting Practice:

  • Upon receipt, divide the antibody into small single-use aliquots (10-20 μL).

  • Use sterile microcentrifuge tubes for aliquoting.

  • Label each tube with antibody details, dilution, and date.

• Freeze-Thaw Cycles:

  • Minimize freeze-thaw cycles as they can lead to antibody denaturation and loss of activity.

  • Once thawed, keep the working aliquot at 4°C if it will be used within 1-2 weeks.

• Handling During Experiments:

  • Keep antibodies on ice when in use during experiments.

  • Avoid prolonged exposure to room temperature.

  • Return to appropriate storage immediately after use.

• Diluted Antibody Solutions:

  • Freshly diluted working solutions can be stored at 4°C for up to one week.

  • For longer storage of diluted antibodies, add BSA (0.5-1%) to stabilize the antibody.

• Contamination Prevention:

  • Always use clean pipette tips.

  • Never return unused antibody to the stock tube.

  • Work in a clean environment to prevent contaminants.

• Monitoring Stability:

  • Keep records of antibody performance over time.

  • If signal quality decreases, this may indicate degradation requiring a new aliquot or fresh antibody.

Following these storage and handling guidelines will help ensure consistent performance of NAA16 antibodies across experiments and maximize the value of these research reagents.

How can I troubleshoot non-specific binding when using NAA16 antibodies in immunofluorescence?

Non-specific binding in immunofluorescence experiments with NAA16 antibodies can obscure legitimate signals and lead to misinterpretation of results. Here's a systematic approach to troubleshooting:

• Optimize Fixation Method:

  • Compare different fixation protocols (paraformaldehyde, methanol, acetone) as they can affect epitope accessibility and non-specific binding.

  • For cytosolic proteins like NAA16, 4% paraformaldehyde for 15-20 minutes at room temperature often works well.

• Blocking Optimization:

  • Increase blocking solution concentration (try 5-10% normal serum from the secondary antibody host species).

  • Add 0.1-0.3% Triton X-100 for permeabilization if using paraformaldehyde fixation.

  • Consider adding 1-5% BSA to reduce non-specific binding.

  • Extend blocking time to 1-2 hours at room temperature or overnight at 4°C.

• Antibody Dilution Series:

  • Prepare a dilution series of the NAA16 antibody (e.g., 1:100, 1:200, 1:500, 1:1000).

  • The optimal dilution should provide specific staining with minimal background.

  • For polyclonal NAA16 antibodies, start with manufacturer-recommended dilutions and adjust as needed .

• Secondary Antibody Controls:

  • Include a control sample with secondary antibody only (no primary NAA16 antibody).

  • This helps identify background caused by the secondary antibody.

  • Consider cross-adsorbed secondary antibodies to reduce cross-reactivity.

• Additional Washing Steps:

  • Increase the number and duration of washes between antibody incubations.

  • Use gentle agitation during washing steps to enhance removal of unbound antibodies.

• Pre-adsorption:

  • Pre-adsorb the NAA16 antibody with cell/tissue lysate from a source that doesn't express NAA16 (if available).

  • This can reduce non-specific binding to common cellular components.

• Signal Amplification Alternatives:

  • If direct detection yields weak signals, consider tyramide signal amplification (TSA).

  • This allows more dilute antibody use while maintaining signal strength.

• Autofluorescence Reduction:

  • Treat samples with sodium borohydride (NaBH₄) briefly before blocking to reduce autofluorescence.

  • Include an unstained sample to identify autofluorescence patterns distinct from antibody staining.

• Peptide Competition:

  • Perform parallel staining with NAA16 antibody pre-incubated with the immunizing peptide.

  • Specific staining should be blocked in this control.

Implementing these approaches systematically will help identify the source of non-specific binding and establish optimal conditions for specific detection of NAA16 in immunofluorescence applications.

How can I use NAA16 antibodies to study protein interactions within the NatA complex?

Studying protein interactions within the NatA complex using NAA16 antibodies requires approaches that preserve native protein complexes:

• Co-Immunoprecipitation (Co-IP):

  • Lyse cells under gentle conditions that preserve protein-protein interactions (e.g., NP-40 or Triton X-100 based buffers).

  • Incubate lysate with NAA16 antibody bound to protein A/G beads overnight at 4°C.

  • Wash thoroughly and elute bound complexes for analysis.

  • Analyze by Western blot for known NatA components (e.g., NAA10) and potential interactors.

  • Include appropriate controls: IgG control, input sample, and if possible, NAA16 knockdown sample.

• Proximity Ligation Assay (PLA):

  • This technique visualizes protein interactions that occur within 40 nm distance in situ.

  • Use NAA16 antibody alongside antibodies against suspected interactors (e.g., NAA10).

  • PLA signals appear as fluorescent dots where interactions occur, enabling spatial analysis of NAA complex formation.

• Immunofluorescence Colocalization:

  • Perform dual immunofluorescence with NAA16 antibody and antibodies against other NatA components.

  • Analyze colocalization using confocal microscopy and quantitative colocalization analysis.

  • Calculate Pearson's or Mander's correlation coefficients to quantify degree of colocalization.

• FRET (Förster Resonance Energy Transfer):

  • Use secondary antibodies labeled with appropriate FRET pairs.

  • FRET occurs only when proteins are in very close proximity (<10 nm), providing evidence for direct interaction.

• Size Exclusion Chromatography coupled with Western Blotting:

  • Fractionate cell lysates by size exclusion chromatography.

  • Analyze fractions by Western blot using the NAA16 antibody.

  • Compare elution profiles with other NatA components to identify complex formation.

• Cross-linking followed by Immunoprecipitation:

  • Treat cells with protein cross-linkers before lysis.

  • Perform immunoprecipitation with NAA16 antibody.

  • Analyze by Western blot or mass spectrometry to identify interacting proteins.

  • Cross-linking captures transient or weak interactions that might be lost during conventional Co-IP.

• BioID or APEX Proximity Labeling:

  • Generate NAA16 fusion proteins with BioID or APEX tags.

  • After biotin labeling, use NAA16 antibodies to confirm expression/localization of the fusion protein.

  • Compare interaction partners identified by proximity labeling with those found using direct immunoprecipitation.

These methods provide complementary information about NAA16 interactions, with each offering different advantages in terms of sensitivity, specificity, and ability to detect transient versus stable interactions within the NatA complex.

What methods can I use to study the impact of NAA16 on N-terminal protein acetylation?

Investigating the role of NAA16 in N-terminal protein acetylation requires techniques that can detect changes in protein acetylation status:

• NAA16 Knockdown/Knockout and N-terminal Proteomics:

  • Utilize siRNA (like siNAA16) to achieve significant depletion of NAA16 expression .

  • Apply N-terminal proteomics with differential stable isotope labeling by amino acids in cell culture (SILAC) and in-vitro 13C 2D3-acetylation .

  • Compare the N-terminal acetylome between control and NAA16-depleted cells.

  • This approach allows identification of specific N-terminal acetylation events dependent on NAA16.

• Western Blotting with Acetylation-Specific Antibodies:

  • Use acetylated lysine antibodies in conjunction with NAA16 antibodies.

  • Compare acetylation levels of specific proteins in control versus NAA16-depleted cells.

  • Useful for studying acetylation of individual proteins of interest.

• Mass Spectrometry-Based Approaches:

  • Employ liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis after NAA16 manipulation.

  • Quantify changes in N-terminal peptide acetylation states.

  • Use SILAC or TMT labeling for quantitative comparisons between experimental conditions.

  • This method provides comprehensive, unbiased analysis of acetylation changes.

• Reporter Protein Systems:

  • Design reporter proteins with N-termini matching known NatA substrates.

  • Express in control and NAA16-manipulated cells.

  • Analyze acetylation status by immunoprecipitation followed by Western blotting or mass spectrometry.

• Pulse-Chase Experiments:

  • Combine NAA16 antibodies with pulse-chase labeling to study the dynamics of protein acetylation and its effects on protein stability.

  • This approach helps distinguish direct acetylation effects from secondary consequences.

• In Vitro Acetylation Assays:

  • Purify the NatA complex with and without NAA16.

  • Perform in vitro acetylation reactions with synthetic peptide substrates.

  • Analyze acetylation by mass spectrometry or HPLC.

  • This approach directly tests the influence of NAA16 on NatA catalytic activity.

• Analysis of Substrate Specificity:

  • Based on N-terminal proteomics data, analyze substrate specificity patterns (see Table 1) :

This multi-method approach allows comprehensive characterization of NAA16's role in N-terminal acetylation, from global proteome effects to mechanistic insights at the molecular level.

How do I interpret conflicting results obtained with different NAA16 antibodies?

Conflicting results with different NAA16 antibodies can be challenging but offer an opportunity to gain deeper insights through systematic analysis:

• Epitope Mapping Analysis:

  • Determine the epitopes recognized by each antibody (check manufacturer information).

  • Antibodies targeting different regions of NAA16 may give different results if:

    • Post-translational modifications mask specific epitopes

    • Protein interactions conceal certain regions

    • Alternative splicing affects epitope presence

• Validation Status Assessment:

  • Review the validation data for each antibody.

  • Well-validated antibodies (with knockout/knockdown controls, peptide competition assays, etc.) should be given more weight .

  • Check if validation was performed in systems similar to yours.

• Isoform-Specific Recognition:

  • Determine if the antibodies might recognize different isoforms of NAA16.

  • Some antibodies might be isoform-specific while others detect all variants.

  • Confirm which isoforms are expressed in your experimental system using RT-PCR.

• Cross-Reactivity Analysis:

  • Test if the antibodies cross-react with NAA15 (the paralog of NAA16).

  • Perform parallel experiments with NAA15 knockdown/overexpression.

  • High sequence similarity between these proteins can lead to cross-reactivity.

• Method-Dependent Efficacy:

  • Some antibodies work well for Western blot but poorly for immunoprecipitation or immunofluorescence.

  • Compare results within each application rather than across different techniques.

  • Optimized protocols for each antibody and application may resolve apparent conflicts.

• Quantification Approaches:

  • Use multiple quantification methods for Western blots (total band intensity, ratio to loading control).

  • Ensure linear range detection for each antibody.

  • Confirm trends with orthogonal methods (e.g., qPCR for expression level changes).

• Experimental Design Solutions:

  • Include additional controls (knockout/knockdown, overexpression).

  • Use alternative detection methods (tagged proteins, mass spectrometry).

  • Perform rescue experiments to confirm specificity of observed effects.

• Reconciliation Strategies:

  • Look for patterns among subsets of antibodies (e.g., do all monoclonals show one result and all polyclonals another?).

  • Consider if differences reflect biologically meaningful phenomena rather than technical artifacts.

  • Use combinatorial approaches (e.g., dual labeling with different antibodies) to directly compare detection patterns.

• Documentation and Reporting:

  • Document all antibodies used (catalog number, lot, dilution, incubation conditions).

  • Report all results transparently, including conflicting ones.

  • Discuss possible explanations for discrepancies based on systematic analysis.

This comprehensive approach treats conflicting results as a scientific opportunity rather than merely a technical problem, potentially revealing new insights about NAA16 biology.

How can NAA16 antibodies be used in conjunction with genome editing techniques?

Combining NAA16 antibodies with genome editing approaches creates powerful experimental systems:

• CRISPR-Cas9 Knockout Validation:

  • Generate NAA16 knockout cell lines using CRISPR-Cas9.

  • Use NAA16 antibodies to confirm complete protein elimination.

  • These validated knockout cells provide excellent negative controls for antibody specificity testing .

  • They also serve as model systems for studying NAA16 loss-of-function effects.

• Knock-in of Tagged NAA16:

  • Use CRISPR-Cas9 to add epitope tags to endogenous NAA16.

  • Compare detection of tagged NAA16 using both tag antibodies and NAA16 antibodies.

  • This approach validates NAA16 antibody specificity while maintaining endogenous expression levels.

• Domain Mapping Studies:

  • Create CRISPR-edited cell lines expressing truncated NAA16 variants.

  • Use domain-specific NAA16 antibodies to map functional regions.

  • This approach provides insights into structure-function relationships.

• RNA-Guided Transcriptional Modulation:

  • Use CRISPRa (activation) or CRISPRi (interference) to modulate NAA16 expression.

  • Quantify expression changes using NAA16 antibodies.

  • Correlate expression levels with functional readouts to establish dose-response relationships.

• Paralog Studies:

  • Generate single and double knockout cells for NAA16 and its paralog NAA15.

  • Use specific antibodies to confirm knockout and examine compensatory expression changes.

  • This approach reveals functional redundancy and specificity between paralogs.

• Modification of Regulatory Elements:

  • Edit NAA16 promoter or enhancer elements using CRISPR-Cas9.

  • Measure resulting changes in protein expression using NAA16 antibodies.

  • This strategy links regulatory genome elements to protein expression outcomes.

• Engineered Cell Systems for Interaction Studies:

  • Edit potential interaction partners of NAA16 (e.g., NAA10) to test interaction requirements.

  • Use co-immunoprecipitation with NAA16 antibodies to assess how mutations affect complex formation.

  • This approach dissects molecular requirements for NatA complex assembly.

• Rescue Experiments:

  • Reintroduce wild-type or mutant NAA16 into knockout cells.

  • Use NAA16 antibodies to confirm expression levels.

  • Measure restoration of function (e.g., N-terminal acetylation patterns).

  • This confirms phenotype specificity and maps critical functional domains.

These integrated approaches leverage the specificity of NAA16 antibodies alongside the precision of genome editing to create powerful experimental systems for studying NAA16 biology at the molecular level.

What proteomics approaches can be combined with NAA16 antibodies for studying the acetylome?

Integrating NAA16 antibodies with proteomics creates powerful approaches for comprehensive acetylome analysis:

• Immunoprecipitation-Mass Spectrometry (IP-MS):

  • Use NAA16 antibodies to immunoprecipitate NAA16 and associated proteins.

  • Analyze by mass spectrometry to identify NatA complex components and novel interactors.

  • This approach can reveal the composition of different NAA16-containing complexes under various conditions.

• N-Terminal COFRADIC (COmbined FRActional DIagonal Chromatography):

  • Compare N-terminal peptides from control and NAA16-manipulated samples.

  • Use NAA16 antibodies to validate knockdown/knockout efficiency.

  • COFRADIC specifically enriches for N-terminal peptides, enabling comprehensive acetylation profiling.

• SILAC-Based Quantitative Proteomics:

  • Label control and NAA16-depleted cells with different isotopes (as in the study described) .

  • Combine samples and analyze by mass spectrometry.

  • This provides precise quantification of acetylation changes dependent on NAA16.

  • The approach revealed that approximately 25% of putative NatA substrates were affected by NatA knockdown .

• Proximity-Dependent Biotin Identification (BioID):

  • Generate BioID-NAA16 fusion proteins.

  • Validate expression using NAA16 antibodies.

  • Identify proteins in proximity to NAA16 through streptavidin pulldown and mass spectrometry.

  • This approach maps the proximal proteome of NAA16, revealing potential new functional connections.

• Acetyl-Lysine Antibody Enrichment Combined with NAA16 Manipulation:

  • Deplete NAA16 using siRNA or CRISPR.

  • Enrich acetylated peptides using acetyl-lysine antibodies.

  • Compare acetylation profiles between control and NAA16-depleted samples.

  • This approach identifies both N-terminal and internal acetylation events affected by NAA16.

• Targeted Proteomics (PRM/SRM):

  • Develop targeted mass spectrometry assays for specific N-terminal acetylated peptides.

  • Monitor these peptides across conditions with different NAA16 expression/activity.

  • This provides highly sensitive quantification of acetylation on specific substrates of interest.

• Pulse-SILAC for Protein Turnover Analysis:

  • Combine NAA16 manipulation with pulse-SILAC to measure protein turnover rates.

  • Use NAA16 antibodies to confirm knockdown/overexpression.

  • This approach reveals how NAA16-dependent acetylation affects protein stability.

  • Research has shown significant impacts on protein stability of knockdown-affected NatA targets .

• Crosslinking Mass Spectrometry (XL-MS):

  • Crosslink proteins in intact cells.

  • Immunoprecipitate using NAA16 antibodies.

  • Analyze by mass spectrometry to identify crosslinked peptides.

  • This technique provides structural information about NAA16-containing complexes.

These integrated proteomics approaches, when combined with NAA16 antibodies for validation and characterization, provide comprehensive insights into the composition, dynamics, and functional impacts of NAA16-containing complexes on the cellular acetylome.