ATAT1 Antibody, HRP conjugated

What is ATAT1 and what are its primary biological functions?

ATAT1 (Alpha-tubulin N-acetyltransferase 1) is an enzyme that catalyzes the acetylation of alpha-tubulin at lysine 40. It plays essential roles in multiple cellular processes across diverse organisms. ATAT1 promotes directional cell locomotion and chemotaxis through AP2A2-dependent acetylation of alpha-tubulin at clathrin-coated pits that concentrate at the leading edge of migrating cells . This process is crucial for controlled cell movement.

The enzyme demonstrates a unique characteristic: it acetylates alpha-tubulin with a relatively slow enzymatic rate due to its catalytic site not being optimized for acetyl transfer. ATAT1 enters microtubules through each end and diffuses throughout the lumen. Due to this slow acetylation rate, it primarily acetylates long-lived/stable microtubules rather than dynamically unstable ones, as it requires sufficient interaction time to execute its function .

Additionally, ATAT1 is essential for normal sperm flagellar function and may facilitate primary cilium assembly. Beyond its acetylation activity, research indicates that ATAT1 regulates forebrain development, as evidenced by studies in knockout mouse models that exhibited ventricular dilation and other developmental abnormalities .

How do ATAT1 antibodies differ in their applications compared to other tubulin-modifying enzyme antibodies?

ATAT1 antibodies are specifically designed to detect the enzyme responsible for alpha-tubulin acetylation, distinguishing it from other tubulin-modifying enzymes like deacetylases (e.g., HDAC6). Unlike antibodies targeting deacetylases that primarily localize to the cytosol, ATAT1 antibodies detect a protein enriched in vesicular fractions, as demonstrated by subcellular fractionation studies .

In experimental contexts, ATAT1 antibodies reveal a distinct punctate distribution along axons that partially overlaps with vesicular markers like BDNF (brain-derived neurotrophic factor), LAMP1 (lysosomal marker), and synaptic vesicle precursors (SV2C and synaptophysin) . This contrasts with the more diffuse cytosolic distribution observed with HDAC6 antibodies. Western blot analysis of subcellular fractions confirms that ATAT1 is selectively enriched in vesicular fractions (P3), while HDAC6 predominantly distributes in cytosolic fractions (S3) .

When designing experiments using different tubulin-modifying enzyme antibodies, researchers should consider these distinct subcellular localizations to properly interpret their results and avoid cross-reactivity issues. The unique vesicular association of ATAT1 makes its antibodies particularly valuable for studying vesicular transport mechanisms and the spatial regulation of tubulin acetylation.

What is the significance of HRP conjugation for ATAT1 antibodies in research applications?

HRP (horseradish peroxidase) conjugation of ATAT1 antibodies provides significant advantages for sensitive detection in multiple experimental applications. The enzyme conjugation eliminates the need for secondary antibody incubation steps, streamlining experimental workflows and reducing potential sources of background noise or cross-reactivity.

In ELISA applications, HRP-conjugated ATAT1 antibodies allow for direct detection through colorimetric, chemiluminescent, or fluorescent substrates. This direct detection system is particularly advantageous when studying ATAT1's complex interactions with microtubules and vesicular components, as it reduces the risk of non-specific binding that can occur with multi-step detection protocols .

For in vitro alpha-tubulin acetylation assays, HRP-conjugated antibodies provide a more direct readout of enzymatic activity. These assays typically involve incubation with acetyl-CoA followed by detection of acetylated alpha-tubulin. Using HRP-conjugated antibodies in this context allows for more precise quantification of acetylation levels . The conjugation also facilitates detection in complex biological samples where signal amplification is needed to visualize low-abundance ATAT1 or where spatial resolution is critical, such as in studies examining ATAT1's association with specific subcellular structures.

What are the optimal conditions for using ATAT1 Antibody, HRP conjugated in immunological assays?

The optimal conditions for using ATAT1 Antibody, HRP conjugated vary by application type. For ELISA applications, the recommended dilution typically ranges from 1:500 to 1:2000, though this should be empirically determined for each specific experimental setup . The antibody performs optimally in phosphate-buffered saline (PBS) with 0.05% Tween 20 and 3% BSA as a blocking buffer to minimize non-specific binding .

Temperature and incubation time significantly impact assay performance. For ELISA, incubation at 37°C for 2 hours with gentle shaking (approximately 100 revolutions per minute) yields reliable results . For longer incubations, such as overnight binding steps, 4°C is recommended to preserve antibody activity and specificity .

Storage conditions are crucial for maintaining antibody performance. The ATAT1 Antibody, HRP conjugated should be stored at -20°C for short-term (weeks to months) or -80°C for long-term storage (months to years). Repeated freeze-thaw cycles should be avoided as they can degrade both the antibody and the HRP conjugate . The antibody is typically supplied in a buffer containing 0.03% Proclin 300, 50% Glycerol, and 0.01M PBS at pH 7.4, which helps maintain stability during storage .

When designing experiments, researchers should include appropriate positive controls using cells known to express ATAT1, such as A549, U-251, THP-1, or U-87 MG cell lines, which have been validated for ATAT1 detection .

How should researchers validate the specificity of ATAT1 antibodies in their experimental models?

Validating ATAT1 antibody specificity requires a multi-faceted approach to ensure reliable experimental results. The gold standard for antibody validation is utilizing knockout (KO) or knockdown (KD) models as negative controls. Several studies have successfully employed ATAT1 knockout mice or knockdown approaches using short hairpin RNA against ATAT1 (shAtat1) . In these models, the absence or significant reduction of signal compared to wild-type samples confirms antibody specificity.

Additionally, researchers should conduct Western blot analyses to verify that the antibody detects a protein of the expected molecular weight for ATAT1 (approximately 42 kDa for the full-length protein). Multiple isoforms of ATAT1 have been identified, with isoform 4 being commonly studied, ranging from amino acids 1-333 . Testing the antibody across different cell lines with known ATAT1 expression patterns, such as A549, U-251, THP-1, and U-87 MG cells, provides further validation of specificity .

For immunohistochemistry or immunofluorescence applications, comparative analysis between tissues known to express ATAT1 (such as testis tissue) and those with minimal expression provides additional validation. When examining brain tissues, researchers should be aware that while ATAT1 knockout models show undetectable tubulin acetylation in most tissues, residual acetylation persists in specific tissues including the heart, skeletal muscle, thymus, and spleen, suggesting the existence of additional α-tubulin acetyltransferases .

Cross-reactivity testing against related proteins, particularly other acetyltransferases, is advisable to confirm specificity. Peptide competition assays, where the antibody is pre-incubated with the immunizing peptide before application to samples, can further confirm binding specificity.

What experimental controls are essential when studying ATAT1-mediated tubulin acetylation?

Robust experimental controls are critical when investigating ATAT1-mediated tubulin acetylation. A comprehensive control strategy includes:

• Genetic controls: Include wild-type (WT) samples alongside ATAT1 knockout (KO) or knockdown (KD) samples. This genetic approach provides the most definitive control for ATAT1-specific effects. Studies have successfully employed 4OH-tamoxifen (4OHT)–inducible Cre systems with Cre-inducible short hairpin RNA against Atat1 (shAtat1) for controlled knockdown .

• Acetylation substrate controls: Since ATAT1 specifically acetylates α-tubulin at lysine 40, controls should include measurements of both acetylated α-tubulin and total α-tubulin levels. The ratio of acetylated α-tubulin to total tubulin provides a normalized measure of acetylation activity . When performing in vitro acetylation assays, include samples with and without acetyl-CoA, the essential cofactor for acetyltransferase activity.

• Catalytic site mutants: Using ATAT1 constructs with mutations in the catalytic domain provides valuable controls to distinguish between acetylation-dependent and acetylation-independent functions of ATAT1. Studies have utilized various truncated ATAT1 forms, including constructs containing only the minimal catalytic domain (amino acids 1-196) and versions lacking portions between this domain and the C-terminal part (amino acids 1-333) .

• Inhibitor controls: Include specific ATAT1 inhibitors alongside vehicle controls to confirm the enzymatic contribution to observed phenotypes. When studying cellular effects, parallel treatment with tubulin-stabilizing agents like Taxol can help differentiate effects due to acetylation versus those due to altered microtubule dynamics .

• Localization controls: Given ATAT1's vesicular enrichment, controls should address whether observed effects stem from ATAT1's direct acetylation activity or its association with vesicular transport. Comparing ATAT1 with its deacetylase counterpart HDAC6, which shows predominantly cytosolic distribution, provides insight into the specificity of vesicular-associated acetylation .

For quantitative analyses, standardized methods for measuring MT acetylation levels should be employed, extracting mean intensity levels of acetylated α-tubulin and total α-tubulin, with background subtraction and calculation of their ratio .

How can ATAT1 antibodies be utilized to investigate the relationship between vesicular transport and microtubule acetylation?

ATAT1 antibodies provide powerful tools for investigating the intricate relationship between vesicular transport and microtubule acetylation. Research has revealed that ATAT1 exhibits a punctate distribution along axons that partially overlaps with vesicular markers such as brain-derived neurotrophic factor (BDNF), suggesting vesicular enrichment of ATAT1 . This localization pattern can be visualized using superresolution microscopy in combination with fluorescently tagged markers.

For studying dynamic transport processes, researchers can employ time-lapse imaging of neurons expressing fluorescently labeled ATAT1 (e.g., ATAT1-GFP) alongside vesicular markers such as LysoTracker or BDNF-mCherry. These studies have demonstrated that ATAT1-GFP clusters move bidirectionally with lysosomes and dense core vesicles at velocities consistent with axonal transport mechanisms (typically 0.1-2 μm/s) . When designing such experiments, researchers should use microfluidic devices that allow for the isolation and visualization of axonal compartments, facilitating the specific analysis of axonal transport processes .

For biochemical characterization, subcellular fractionation coupled with western blot analysis using ATAT1 antibodies can confirm the association of ATAT1 with vesicular fractions. This technique has revealed that ATAT1 is selectively enriched in the vesicular fraction (P3), contrasting with the predominant cytosolic distribution of deacetylases like HDAC6 (S3) . Furthermore, proteomic analysis of vesicular fractions using mass spectrometry can identify co-transported proteins, with studies detecting ATAT1 together with kinesins and dyneins in vesicular proteomic content .

To directly assess the functional relationship between vesicular ATAT1 and microtubule acetylation, in vitro reconstitution assays can be performed. Purified vesicles from mouse brains can be labeled with fluorescent tracers and loaded into flow chambers containing stabilized microtubules. The addition of acetyl-CoA allows for real-time visualization of acetylation events using TIRF microscopy, followed by immunofluorescence analysis with acetylated α-tubulin antibody to quantify microtubule acetylation levels .

What methodologies can assess ATAT1's role in axonal transport and neuronal development?

Investigating ATAT1's role in axonal transport and neuronal development requires integrating multiple experimental approaches. Time-lapse imaging provides direct visualization of transport dynamics affected by ATAT1 manipulation. Researchers can perform live recordings of organotypic brain slices from wild-type and ATAT1 knockout or knockdown models, tracking fluorescently labeled organelles such as Lamp1-Emerald-positive lysosomes . These experiments have revealed that ATAT1 loss significantly impairs both anterograde and retrograde axonal transports, reducing average and instantaneous velocities, decreasing run lengths, and increasing pausing time of organelles .

For more controlled experimental conditions, primary cortical neurons can be cultured in microfluidic devices that physically separate axons from cell bodies. Fluorescent probes for lysosomes and mitochondria allow for detailed analysis of organelle movement parameters. Studies employing this approach have confirmed transport deficits in ATAT1 knockout neurons, with quantifiable changes in velocity, directionality, and pausing behavior .

To examine ATAT1's developmental roles, in utero electroporation (IUE) techniques can be utilized to manipulate ATAT1 expression in developing cortical neurons. This approach allows for temporal control of gene knockdown or overexpression and subsequent analysis of neuronal migration, axon outgrowth, and connectivity. Conditional knockout strategies, using inducible Cre-loxP systems, provide another method for studying ATAT1's function during specific developmental windows .

For examining cellular architecture, researchers can use immunohistochemistry with antibodies against acetylated α-tubulin alongside ATAT1 antibodies to correlate acetylation levels with developmental phenotypes. This approach has revealed that ATAT1 knockout mice exhibit ventricular dilation and developmental abnormalities in the forebrain, including alterations in the lateral ventricle, septum, and striatum .

Quantitative analysis of transport parameters should follow standardized protocols, with vesicles considered stationary if speed is lower than 0.1 μm/s . For developmental phenotyping, comprehensive histological analysis combined with behavioral testing provides insights into the functional consequences of ATAT1 deficiency.

How does ATAT1 contribute to stress-induced tubulin hyperacetylation and what experimental approaches can test this relationship?

ATAT1 plays a critical role in stress-induced tubulin hyperacetylation, serving as a cellular response mechanism to various environmental stressors. To investigate this relationship, researchers can employ several sophisticated experimental approaches:

Stress induction protocols can be designed to examine ATAT1's response to different stressors. Cultured cells or model organisms can be subjected to controlled stressors such as heat shock, oxidative stress (H₂O₂ treatment), or chemical stressors. Following stress exposure, researchers can quantify changes in tubulin acetylation levels using acetylated α-tubulin antibodies in conjunction with ATAT1 antibodies to establish correlation or causation .

Genetic manipulation approaches provide direct evidence of ATAT1's involvement in stress responses. ATAT1 knockout mice have been instrumental in demonstrating that ATAT1 governs tubulin hyperacetylation during stress responses . For temporal control of ATAT1 activity, inducible expression systems or rapid protein degradation approaches (such as auxin-inducible degron systems) can be employed to manipulate ATAT1 levels immediately before or during stress exposure.

For biochemical characterization of stress-mediated changes in ATAT1 activity, in vitro α-tubulin acetylation assays can be performed using cell extracts from stressed and unstressed conditions. These assays typically involve incubation with acetyl-CoA, followed by detection with acetylated α-tubulin antibody and peroxidase-conjugated secondary antibodies . Mass spectrometry analysis can further identify post-translational modifications on ATAT1 that might regulate its activity during stress responses, as demonstrated in studies using S-Trap columns for sample preparation followed by LC/MS analysis .

Live-cell imaging using fluorescently tagged ATAT1 allows real-time visualization of ATAT1 redistribution during stress responses. This approach can be combined with photoactivatable or photoconvertible tubulin to track newly acetylated microtubule populations formed during stress recovery. Super-resolution microscopy techniques like TIRF (Total Internal Reflection Fluorescence) microscopy provide the spatial resolution necessary to visualize ATAT1's interaction with microtubules during stress responses .

The relationship between stress-induced acetylation and cellular resilience can be assessed through viability assays comparing wild-type and ATAT1-deficient cells following stress exposure, establishing functional consequences of ATAT1-mediated tubulin modifications in stress adaptation.

What are common technical challenges when using ATAT1 antibodies and how can they be addressed?

Researchers frequently encounter several technical challenges when working with ATAT1 antibodies. Background signal issues are common in immunodetection applications. To address this, optimization of blocking conditions is essential—using 3-5% BSA in PBS with 0.05% Tween 20 typically provides effective blocking . For tissue sections, extending blocking time to 1-2 hours at room temperature can significantly reduce background. Additionally, titrating antibody dilutions (starting with manufacturer recommendations of 1:500-1:2000 for WB and 1:50-1:500 for IHC/IF) helps determine the optimal signal-to-noise ratio for each experimental system .

Cross-reactivity with other acetyltransferases may occasionally occur. This can be mitigated by using ATAT1 knockout or knockdown samples as negative controls to confirm antibody specificity . Peptide competition assays, where the antibody is pre-incubated with the immunizing peptide (in this case, recombinant Human Alpha-tubulin N-acetyltransferase 1 protein 194-238AA), can also help distinguish specific from non-specific binding .

Detecting endogenous ATAT1 can be challenging due to potentially low expression levels in some tissues. For immunohistochemistry applications, optimizing antigen retrieval methods is crucial. The recommended approach is TE buffer pH 9.0, although citrate buffer pH 6.0 may alternatively be used . For particularly difficult samples, signal amplification systems compatible with HRP conjugates can enhance detection sensitivity.

Batch-to-batch variability in antibody performance may occur. Researchers should validate each new lot against previously successful lots using positive control samples such as A549, U-251, THP-1, or U-87 MG cells, which are known to express ATAT1 . Additionally, storing antibody aliquots at -80°C for long-term storage rather than repeatedly freezing and thawing the stock helps maintain consistent performance .

For optimal results in specialized applications, researchers should consider testing multiple fixation protocols. For immunofluorescence of microtubules, methanol fixation at -20°C for 10 minutes has proven effective for preserving microtubule structures while allowing antibody access to epitopes .

How should researchers interpret discrepancies between ATAT1 protein levels and tubulin acetylation patterns?

Interpreting discrepancies between ATAT1 protein levels and tubulin acetylation patterns requires careful consideration of several biological mechanisms. First, researchers should recognize that ATAT1's catalytic rate is inherently slow, meaning that even high ATAT1 protein levels may not immediately translate to elevated tubulin acetylation . The enzyme primarily acetylates long-lived/stable microtubules due to this slow rate, as it requires sufficient time to act on microtubules before they undergo dynamic instability .

Multiple regulatory factors influence ATAT1 activity beyond protein levels. Post-translational modifications of ATAT1 itself may alter its enzymatic efficiency without changing protein abundance. Additionally, the availability of acetyl-CoA, ATAT1's essential cofactor, may limit acetylation reactions even when enzyme levels are high. Subcellular compartmentalization also plays a crucial role—ATAT1 must access the microtubule lumen to acetylate α-tubulin at lysine 40, and barriers to this access could create discrepancies between enzyme levels and acetylation patterns .

Importantly, research has revealed that ATAT1 is not the only enzyme capable of acetylating α-tubulin. Studies in ATAT1 knockout mice have demonstrated residual tubulin acetylation in specific tissues including the heart, skeletal muscle, trachea, oviduct, thymus, and spleen, strongly suggesting the existence of additional α-tubulin acetyltransferases . This finding explains why some tissues may maintain acetylation patterns despite ATAT1 deficiency.

The balance between acetylation and deacetylation activities further complicates interpretation. HDAC6 and SIRT2 are major tubulin deacetylases that counteract ATAT1's activity. Changes in these deacetylases' expression or activity could significantly alter acetylation patterns independent of ATAT1 levels. Unlike ATAT1, which shows vesicular enrichment, HDAC6 predominantly localizes to the cytosol, creating spatial regulation of the acetylation/deacetylation balance .

When designing experiments to investigate these discrepancies, researchers should employ multiple methodologies including acetylation site-specific antibodies, activity assays for both ATAT1 and tubulin deacetylases, and analysis of microtubule dynamics to comprehensively understand the relationship between enzyme levels and functional outcomes.

What considerations are important when integrating ATAT1 antibody data with functional studies of tubulin acetylation?

Integrating ATAT1 antibody data with functional studies of tubulin acetylation requires careful attention to several methodological considerations. Temporal dynamics are crucial—ATAT1-mediated acetylation occurs at a relatively slow rate, primarily affecting long-lived/stable microtubules rather than dynamically unstable ones . Consequently, short-term manipulations of ATAT1 levels may not immediately alter acetylation patterns. Experimental timelines should allow sufficient duration for acetylation changes to manifest, particularly when studying effects on cellular processes like migration or axonal transport.

Quantification methods significantly impact data interpretation. When measuring acetylation levels, researchers should normalize acetylated α-tubulin to total α-tubulin rather than using absolute acetylation values, accounting for potential differences in total microtubule content between samples . For imaging-based quantification, standardized protocols should extract mean intensity levels across defined regions (e.g., entire axons or full width of motoneurons), with proper background subtraction .

The relationship between ATAT1's dual roles must be considered: it functions both as an enzyme that acetylates microtubules and as a component of vesicular transport. Studies have shown that ATAT1 colocalizes with vesicular markers and moves bidirectionally with lysosomes and dense core vesicles . This dual functionality means that phenotypes observed following ATAT1 manipulation might result from altered enzymatic activity, disrupted vesicular transport, or both. To distinguish between these possibilities, complementary approaches such as rescue experiments with catalytically inactive ATAT1 can be informative.

Technical aspects of antibody application affect data quality. For functional studies integrating ATAT1 antibodies with live-cell imaging, researchers must ensure that antibody binding doesn't interfere with ATAT1's normal function or localization. For fixed samples, different fixation methods may expose different epitopes, potentially leading to varying detection efficiency. Methanol fixation at -20°C for 10 minutes has been validated for preserving microtubule structures while allowing antibody access to epitopes .

Considering the cellular context is essential. ATAT1 function varies across cell types and developmental stages. For instance, ATAT1 knockout mice show severe forebrain developmental abnormalities but maintain a normal hematopoietic system . This context-dependent functionality must be accounted for when extrapolating findings across different experimental systems.