ATAT1 Monoclonal Antibody

What epitopes do commercial ATAT1 monoclonal antibodies typically recognize?

Commercial ATAT1 monoclonal antibodies, such as clone N347/42, typically recognize the N-acetyltransferase domain of ATAT1. This domain corresponds to amino acids 2-236 of human ATAT1 (accession number Q5SQI0) and is highly conserved across species, enabling cross-reactivity between human, mouse, and rat samples . When selecting an antibody for your research, consider whether the epitope might be masked by protein-protein interactions or post-translational modifications in your specific experimental context.

What molecular weight band should researchers expect when using ATAT1 antibodies in Western blot?

When using ATAT1 antibodies for Western blot applications, researchers should expect to detect a band at approximately 40-45 kDa . This corresponds to the calculated molecular weight of ATAT1, which is reported to be around 47 kDa . Multiple isoforms of ATAT1 have been reported , which may contribute to slight variations in observed molecular weights across different tissues and experimental conditions.

What is the species reactivity profile of ATAT1 monoclonal antibodies?

Most commercially available ATAT1 monoclonal antibodies demonstrate reactivity with human, mouse, and rat samples . This cross-reactivity is expected due to the high conservation of the N-acetyltransferase domain across mammalian species. For example, the N347/42 clone has been validated specifically for these three species . When planning experiments with other organisms, preliminary validation is essential despite sequence homology predictions.

How do researchers validate the specificity of ATAT1 monoclonal antibodies?

To ensure ATAT1 antibody specificity, researchers should employ multiple validation strategies:

• Genetic validation using ATAT1 knockout samples - The most definitive approach is comparing antibody reactivity in wild-type versus ATAT1 knockout tissues or cells

• Knockdown validation - Testing antibody reactivity following siRNA-mediated ATAT1 depletion

• Expression pattern analysis - Verifying expected tissue distribution patterns, with highest expression in brain, testis, kidney, and gastrointestinal tract

• Multiple antibody comparison - Comparing staining patterns using antibodies targeting different ATAT1 epitopes

• Positive control testing - Confirming reactivity in cell lines with validated ATAT1 expression (A549, U-251, THP-1, U-87 MG cells)

What are the typical storage and handling conditions for ATAT1 monoclonal antibodies?

ATAT1 monoclonal antibodies are typically stored in buffered solutions containing preservatives. For example:

• Storage buffer: 10 mM Tris, 50 mM Sodium Chloride, 0.065% Sodium Azide, pH 7.125 or PBS with 0.02% sodium azide and 50% glycerol, pH 7.3

• Storage temperature: -20°C for long-term storage

• Aliquoting: Recommended to avoid repeated freeze-thaw cycles

• Centrifugation: Prior to opening, centrifuge the vial for maximum recovery

• Stability: Typically stable for up to 24 months from date of receipt when properly stored

What are the optimal applications for ATAT1 monoclonal antibodies?

ATAT1 monoclonal antibodies have been validated for multiple research applications:

For studying ATAT1's dynamic subcellular localization, which significantly impacts its function, IF/ICC applications are particularly valuable .

What tissue preparation techniques are optimal for ATAT1 immunohistochemistry?

For successful ATAT1 immunohistochemistry:

• Fixation: 4% paraformaldehyde is typically used for brain and other tissues

• Antigen retrieval: TE buffer at pH 9.0 is recommended as the primary method, with citrate buffer at pH 6.0 as an alternative

• Blocking: BSA or normal serum matching the secondary antibody host

• Antibody incubation: Overnight at 4°C at dilutions of 1:50-1:500

• Signal detection: DAB or fluorescent secondary antibodies depending on application

For mouse brain tissues, where ATAT1 plays a critical role in forebrain development , thorough perfusion fixation is essential for consistent staining patterns.

How can researchers optimize ATAT1 detection in Western blot applications?

For optimal Western blot detection of ATAT1:

• Sample preparation:

  • Include protease inhibitors to prevent degradation

  • Add phosphatase inhibitors if studying phosphorylation-dependent localization

  • Use RIPA or NP-40 based lysis buffers for efficient extraction

• Gel and transfer parameters:

  • 10-12% polyacrylamide gels provide optimal resolution for 40-45 kDa ATAT1

  • Semi-dry transfer systems work well with standard PVDF membranes

• Blocking and antibody conditions:

  • Block with 5% non-fat milk or BSA in TBST

  • Use antibody dilutions between 1:500-1:2000

  • Incubate primary antibody overnight at 4°C for best results

• Detection considerations:

  • Both chemiluminescent and fluorescent detection systems are compatible

  • Include positive control lysates from brain tissue or validated cell lines

What controls should be included when using ATAT1 antibodies?

Proper experimental controls are essential for reliable interpretation of ATAT1 antibody results:

• Positive controls:

  • Tissues known to highly express ATAT1 (brain, testis, kidney, retina)

  • Validated cell lines: A549, U-251, THP-1, U-87 MG cells

• Negative controls:

  • ATAT1 knockout tissues/cells when available

  • Isotype control (IgG1 for N347/42 clone)

  • Primary antibody omission controls

• Specificity controls:

  • Peptide competition assays

  • Comparison with alternative ATAT1 antibodies

  • Correlation with mRNA expression data

• Experimental condition controls:

  • Wild-type controls for stress experiments that induce tubulin acetylation

  • Vehicle controls for drug treatments that might affect ATAT1 function

How can researchers use ATAT1 antibodies to study subcellular localization changes?

ATAT1 exhibits dynamic intracellular localization that significantly impacts its function . To study this:

• Immunofluorescence approach:

  • Use ATAT1 monoclonal antibodies at 1:50-1:500 dilution

  • Co-stain with subcellular markers (nuclear, cytoskeletal, etc.)

  • Include DAPI or similar nuclear counterstain

  • Use confocal microscopy for precise localization determination

• Quantification methods:

  • Calculate nuclear/cytosolic ratios as described in research

  • Apply colocalization correlation analyses for interaction studies

  • Categorize cells based on ATAT1 distribution patterns (nuclear excluded, diffused, nuclear enriched)

• Live-cell imaging alternatives:

  • Compare with fluorescently-tagged ATAT1 constructs like mVenus-α-TAT1

  • Validate that tag doesn't interfere with localization patterns

• Biochemical verification:

  • Complement imaging with subcellular fractionation and Western blotting

  • Compare cytosolic, nuclear, and cytoskeletal fractions

How can ATAT1 antibodies be used to study stress-induced tubulin acetylation?

ATAT1 is essential for stress-induced tubulin hyperacetylation . To investigate this phenomenon:

• Experimental design:

  • Expose cells to stressors like high salt, high glucose, or H₂O₂

  • Include appropriate vehicle controls and time-course analyses

  • Compare wild-type and ATAT1-deficient cells

• Dual immunostaining approach:

  • Use ATAT1 monoclonal antibodies to track localization changes

  • Simultaneously detect acetylated tubulin levels with specific antibodies

  • Analyze colocalization between ATAT1 and acetylated tubulin

• Biochemical analysis:

  • Western blot for both ATAT1 and acetylated tubulin levels

  • Include appropriate loading controls (total tubulin)

  • Quantify acetylation relative to total tubulin levels

• Genetic complementation:

  • Rescue experiments in ATAT1-knockout cells with wild-type or mutant constructs

  • Test structure-function relationships using domain mutants

What methodological approaches help distinguish ATAT1 from other acetyltransferases?

Although ATAT1 is the predominant α-tubulin acetyltransferase in vivo , evidence suggests additional acetyltransferases exist . To distinguish these activities:

• Genetic approaches:

  • Compare acetylation patterns in wild-type versus ATAT1 knockout tissues

  • Focus on tissues with residual acetylation in ATAT1 knockout models (heart, skeletal muscle, thymus, spleen)

  • Combine ATAT1 knockout with knockdown of candidate acetyltransferases

• Biochemical strategies:

  • Immunodepletion of ATAT1 followed by enzymatic activity assays

  • In vitro competition assays with recombinant enzymes

  • Mass spectrometry to identify acetylation sites beyond K40

• Inhibitor studies:

  • Compare effects of pan-acetyltransferase versus specific inhibitors

  • Analyze dose-response relationships in different tissues

• Substrate specificity analysis:

  • Test activity on different tubulin isotypes

  • Examine dependence on tubulin polymerization state

How should researchers address technical challenges when studying ATAT1 in brain tissues?

Brain tissue presents unique challenges for ATAT1 antibody applications:

• Regional expression considerations:

  • ATAT1 is highly expressed throughout the brain with specific enrichment in the septum, striatum, and cerebral cortex

  • The dentate gyrus shows structural differences in ATAT1 knockout mice

  • Different fixation and antigen retrieval protocols may be needed for various brain regions

• Developmental timing factors:

  • ATAT1 regulates forebrain development and neuronal migration

  • Expression patterns change during development, with ventricular enlargement becoming evident at postnatal day 5

  • Age-matched controls are essential for developmental studies

• Technical optimizations:

  • Extended perfusion fixation for consistent penetration

  • Longer antigen retrieval for fixed brain tissues

  • Autofluorescence reduction techniques (Sudan Black B, sodium borohydride)

  • Longer washing steps to reduce background

• Advanced detection approaches:

  • Tyramide signal amplification for low abundance detection

  • Multi-label immunofluorescence to correlate with cell-type markers

  • Serial section analysis for three-dimensional reconstruction

What strategies help researchers investigate ATAT1's role in neuronal development?

ATAT1 plays critical roles in forebrain development and neuronal migration . To investigate these functions:

• Birth-dating experiments:

  • BrdU pulse-chase labeling to track neuronal migration as described in ATAT1 research

  • EdU labeling with click chemistry detection as a non-antibody alternative

• Neuronal migration analysis:

  • Immunostaining for layer-specific markers (Cux1, Cux2, Tbr1, Ctip2)

  • Quantification of neuronal positioning in developing brain regions

  • Time-lapse imaging of neuronal migration in slice cultures

• Proliferation assessment:

  • Ki67 immunostaining for cycling cells

  • BrdU pulse labeling for S-phase analysis

  • Phospho-histone H3 for mitotic cells

• Cellular morphology evaluation:

  • Golgi staining for dendritic complexity analysis

  • DiI labeling for axonal projections

  • Immunostaining for cytoskeletal elements (acetylated tubulin, actin)

• Functional correlates:

  • Behavioral tests like rotarod for motor coordination deficits

  • Electrophysiological recordings for functional connectivity

How can researchers use ATAT1 antibodies to investigate phosphorylation-dependent regulation?

Research indicates that ATAT1 localization and function are regulated by phosphorylation . To investigate this regulatory mechanism:

• Detection approaches:

  • Use antibodies against total ATAT1 alongside phospho-specific antibodies if available

  • Combine with phosphatase treatments to confirm phosphorylation status

  • Employ Phos-tag gels to separate phosphorylated from non-phosphorylated ATAT1

• Subcellular localization analysis:

  • Compare distribution patterns under conditions affecting phosphorylation

  • Quantify nuclear/cytosolic ratios following kinase or phosphatase treatments

  • Co-stain with phospho-motif antibodies for correlation analysis

• Mutational studies:

  • Compare wild-type ATAT1 with phospho-mimetic or phospho-dead mutants

  • Assess effects on localization and acetyltransferase activity

  • Perform rescue experiments in ATAT1-deficient backgrounds

• Signaling pathway investigation:

  • Test effects of kinase inhibitors on ATAT1 localization and function

  • Investigate stress-responsive pathways that might phosphorylate ATAT1

  • Correlate with stress-induced tubulin hyperacetylation

How should researchers interpret differences between in vitro and in vivo findings with ATAT1?

Reconciling in vitro and in vivo observations about ATAT1 requires careful consideration:

• Functional context differences:

  • ATAT1 knockout mice are viable but show specific developmental defects

  • Despite apparent normal development in many tissues, ATAT1 knockout mice show enlarged lateral ventricles and specific defects in the septum and striatum

  • Motor coordination deficits emerge despite largely normal gross development

• Methodological considerations:

  • Cell culture conditions may not recapitulate physiological stress states

  • Immortalized cell lines may have altered tubulin modification patterns

  • Developmental timing and cell-type specific effects are difficult to model in vitro

• Compensatory mechanisms:

  • Residual tubulin acetylation in some tissues of ATAT1 knockout mice suggests additional acetyltransferases

  • Developmental compensation may mask acute effects observed in vitro

  • Redundant mechanisms may ensure basic developmental processes proceed

• Interpretation framework:

  • Focus on specific cellular processes rather than general viability

  • Consider both direct ATAT1 targets and secondary effects of altered microtubule acetylation

  • Evaluate phenotypes in various contexts (development, stress response, aging)

What explains tissue-specific patterns of residual tubulin acetylation in ATAT1 knockout models?

Despite ATAT1 being the predominant α-tubulin acetyltransferase, some tissues retain acetylation in knockout models:

• Tissue-specific patterns:

  • Residual tubulin acetylation detected in heart, skeletal muscle, trachea, oviduct, thymus, and spleen of ATAT1 knockout mice

  • Complete loss of acetylation in most other tissues

• Potential explanations:

  • Tissue-specific expression of alternative acetyltransferases

  • Differential regulation of deacetylases (HDAC6, SIRT2) across tissues

  • Variation in tubulin isotype composition affecting enzyme recognition

  • Tissue-specific protective mechanisms against deacetylation

• Investigative approaches:

  • Comparative proteomics of tissues with and without residual acetylation

  • Transcriptome analysis to identify candidate acetyltransferases

  • Pharmacological inhibition of deacetylases to test stability differences

  • Immunoprecipitation of acetylated tubulin followed by mass spectrometry

• Functional significance:

  • Correlation with tissue-specific phenotypes in ATAT1 knockout models

  • Analysis of whether residual acetylation is sufficient for function

  • Investigation of whether these tissues employ additional stabilization mechanisms

How should researchers interpret dual bands or molecular weight shifts with ATAT1 antibodies?

When encountering unexpected band patterns in ATAT1 Western blots:

• Potential biological explanations:

  • Multiple ATAT1 isoforms have been reported

  • Phosphorylation can alter electrophoretic mobility

  • Proteolytic processing during sample preparation

  • Alternative splicing variants

• Verification approaches:

  • Compare patterns with multiple ATAT1 antibodies targeting different epitopes

  • Include ATAT1 knockout controls to identify specific bands

  • Use phosphatase treatment to determine if phosphorylation causes mobility shifts

  • Employ size fractionation or immunoprecipitation for band identification

• Technical considerations:

  • Sample preparation methods (lysis buffers, protease inhibitors) affect observed patterns

  • Gel percentage and running conditions impact band resolution

  • Transfer efficiency varies for different molecular weight proteins

• Reporting recommendations:

  • Document all observed bands and their relative intensities

  • Specify exact molecular weight markers used

  • Include positive control samples with established band patterns

  • Note sample preparation conditions that might affect banding patterns

What factors explain variability in ATAT1 subcellular localization patterns?

ATAT1 shows variable subcellular distribution that significantly impacts its function :

• Observed patterns:

  • Distinct nuclear exclusion in approximately 80% of cells

  • Diffused patterns or nuclear enrichment in approximately 20% of cells

  • Dynamic changes in response to cellular conditions

• Regulatory mechanisms:

  • Phosphorylation status appears to regulate localization

  • Cell cycle phase may influence distribution patterns

  • Stress conditions can alter ATAT1 localization and activity

• Functional significance:

  • Localization affects accessibility to microtubule substrates

  • Compartmentalization may regulate acetylation of specific microtubule populations

  • Nuclear-cytoplasmic shuttling may coordinate with cellular stress responses

• Methodological considerations:

  • Fixation methods can affect observed localization patterns

  • Expression levels of tagged constructs may influence distribution

  • Live versus fixed cell analysis may yield different results

How should researchers correlate ATAT1 expression with functional outcomes?

To establish meaningful connections between ATAT1 expression and functional consequences:

• Multi-level analysis approach:

  • Combine protein expression (antibody-based detection) with functional readouts

  • Correlate ATAT1 levels with tubulin acetylation patterns

  • Link molecular findings to cellular phenotypes and behavioral outcomes

• Genetic manipulation strategies:

  • Compare wild-type, heterozygous, and homozygous knockout models

  • Use conditional knockouts to assess tissue-specific requirements

  • Employ rescue experiments with wildtype or mutant constructs

• Developmental timing considerations:

  • Track ATAT1 expression and function across developmental stages

  • Note that ventricular enlargement becomes apparent at postnatal day 5

  • Correlate with critical periods of neural migration and circuit formation

• Quantitative approaches:

  • Perform dose-response analyses with varying ATAT1 levels

  • Develop computational models incorporating enzyme kinetics

  • Establish thresholds required for different cellular functions

• Translational perspectives:

  • Correlate findings with human conditions showing ventricular enlargement

  • Consider ATAT1 as a potential modifier gene in neurodevelopmental disorders

  • Investigate stress-protective roles in cellular and organismal contexts