Phospho-TUBA1A/TUBA1B/TUBA1C/TUBA3C/TUBA3E/TUBA4A (Tyr272) Antibody
Product Specs
Target Background
- A de novo heterozygous c.320A>G [p.(His 107 Arg)] mutation in TUBA1A was identified in a patient with microcephaly, epileptic seizures, and severe developmental delay. PMID: 29109381
- Given that Spastin interacts with microtubules in two locations, we propose that severing occurs through forces exerted on the C-terminal tail of tubulin, resulting in a conformational change in tubulin, which releases it from the polymer. PMID: 17389232
- Molecular docking studies indicated that 6f effectively bound to the colchicine-binding site of tubulin. Moreover, 6f treatment induced G2/M cell cycle arrest in a dose-dependent manner and subsequently triggered cell apoptosis. PMID: 28440465
- Induced pluripotent stem cells (iPSCs) were generated from the umbilical cord and peripheral blood of two lissencephaly patients with different clinical severities carrying alpha tubulin (TUBA1A) missense mutations. PMID: 27431206
- Long intergenic non-coding RNA APOC1P1-3 inhibits apoptosis by decreasing alpha-tubulin acetylation in breast cancer. PMID: 27228351
- Results demonstrate that Tuba1a plays a critical, non-compensated role in neuronal saltatory migration in vivo, highlighting the importance of microtubule flexibility in nucleus-centrosome coupling and neuronal-branching regulation during neuronal migration. PMID: 28687665
- Data suggest that TUBA1A mutations disrupting lateral interactions have pronounced dominant-negative effects on microtubule dynamics, which are associated with the severe end of the lissencephaly spectrum. PMID: 26493046
- Data indicate that tubulin phosphorylation and acetylation play significant roles in regulating microtubule assembly and stability. PMID: 26165356
- Data demonstrate that plasma membrane Ca(2+)-ATPase (PMCA) was associated with tubulin in both normotensive and hypertensive erythrocytes. PMID: 26307527
- Studies indicate that alpha-tubulin acetylation and microtubule levels are primarily governed by opposing actions of alpha-tubulin acetyltransferase 1 (ATAT1) and histone deacetylase 6 (HDAC6). PMID: 26227334
- Data from studies using a peptide fragment of alpha-tubulin (residues 31-49) suggest that Ser38 is crucial for substrate recognition by alpha-tubulin acetylase 1 (ATAT1); Asp39, Ile42, the glycine stretch (residues 43-45), and Asp46 are also involved. PMID: 25602620
- Lysine 40 acetylation of alpha-tubulin does not result in significant changes in kinesin-1's landing rate or motility parameters. PMID: 24940781
- These results demonstrated that SelP interacts with tubulin, alpha 1a (TUBA1A). PMID: 24914767
- This study shows that all fetuses with lissencephaly and cerebellar hypoplasia carried distinct TUBA1A mutations. PMID: 25059107
- These findings highlight PKC-mediated phosphorylation of alpha-tubulin as a novel mechanism for controlling the dynamics of microtubules that result in cell movement. PMID: 24574051
- This case provides new insights into the wide spectrum of disease phenotypes associated with TUBA1A mutation. PMID: 23528852
- The present study confirms that mutations in tubulin genes are responsible for complex brain malformation. PMID: 24392928
- Studies suggest that tubulin-interactive agents have the potential to play a significant role in the fight against cancer. PMID: 23818224
- Missense mutations in TUBA1A were found in 3 patients with polymicrogyria. PMID: 22948023
- We described the clinical course and pathological findings in a child with a TUBA1A mutation. PMID: 22633752
- TUBA1A and TUBB2B coding regions have been sequenced that are associated with cortical malformations. PMID: 23361065
- Data show that Na(+),K(+)-ATPase activity was >50% lower and membrane-associated tubulin content was >200% higher in erythrocyte membranes from diabetic patients. PMID: 22565168
- This study describes a 14-month-old girl with TUBA1A mutation-associated lissencephaly, and summarizes the clinical and neuroradiologic findings of 19 cases in the literature. PMID: 22264709
- Alpha2B-adrenergic receptor interaction with tubulin controls its transport from the endoplasmic reticulum to the cell surface. PMID: 21357695
- The expression of alpha-tubulin and MDR1 may play an important role in the development and progression of human non-small cell lung carcinoma. PMID: 20510079
- We report a mutation in TUBA1A as a cause of polymicrogyria. To date, all mutations in TUBA1A have occurred de novo, resulting in isolated cases. This article describes familial recurrence of TUBA1A mutations due to somatic mosaicism in a parent. PMID: 21403111
- Data show that IAV-infected cells contain elevated levels of AcTub and alpha-tubulin. PMID: 21094644
- Mutations in TUBA1A result in defects in tubulin folding and heterodimer assembly. PMID: 20603323
- LIS-associated mutations of TUBA1A operate through diverse mechanisms that include disruption of binding sites for microtubule-associated proteins. PMID: 20466733
- The dipole moments of each tubulin isotype may influence their functional characteristics within the cell, leading to differences in MT assembly kinetics and stability. PMID: 16941085
- Mutations in alpha-tubulin in mice and humans that affect neuronal migration result in abnormal lamination of brain structures with associated behavioral deficits. PMID: 17218254
- Retrospective examination of MR images suggests that patients with TUBA1A mutations share not only cortical dysgenesis, but also cerebellar, hippocampal, corpus callosum, and brainstem abnormalities. PMID: 17584854
- Increased expression of tubulin alpha is associated with pulmonary sclerosing hemangioma. PMID: 17914564
- The diminished production of TUBA1A tubulin in R264C individuals is consistent with haploinsufficiency as a cause of the disease phenotype. PMID: 18199681
- The TUBA1A phenotype is distinct from LIS1, DCX, RELN, and ARX lissencephalies. Compared with the phenotypes of children mutated for TUBA1A, these prenatally diagnosed fetal cases occur at the severe end of the TUBA1A lissencephaly spectrum. PMID: 18669490
- Missense mutations within the TUBA1A gene are associated with specific abnormalities in lissencephaly. PMID: 18728072
- Mutation analysis was conducted in the TUBA1A gene in 46 patients with classical lissencephaly. PMID: 18954413
- This protein has been found to be differentially expressed in the Wernicke's Area from patients with schizophrenia. PMID: 19405953
Q&A
What is the specificity of Phospho-TUBA1A/TUBA1B/TUBA1C/TUBA3C/TUBA3E/TUBA4A (Tyr272) Antibody?
Phospho-TUBA1A/TUBA1B/TUBA1C/TUBA3C/TUBA3E/TUBA4A (Tyr272) antibody specifically recognizes endogenous levels of tubulin alpha proteins only when phosphorylated at tyrosine 272. This phospho-specific antibody is produced by immunizing rabbits with synthetic phosphopeptides derived from human TUBA proteins around the phosphorylation site, using the sequence A-T-Y(p)-A-P. The antibody's specificity is ensured through affinity chromatography purification with the immunizing phosphopeptide, followed by removal of non-phospho-specific antibodies using chromatography with non-phosphopeptides . This high specificity makes it valuable for detecting post-translational modifications in tubulin that may play crucial roles in microtubule dynamics and cellular functions.
What are the recommended experimental applications and dilutions for this antibody?
The Phospho-TUBA1A/TUBA1B/TUBA1C/TUBA3C/TUBA3E/TUBA4A (Tyr272) antibody has been validated for the following applications with recommended dilutions:
For optimal results, researchers should perform antibody titration experiments to determine the ideal concentration for their specific experimental system, as sensitivity may vary depending on expression levels of phosphorylated tubulin in different cell types and conditions .
Tyrosine 272 phosphorylation represents a critical post-translational modification that influences microtubule dynamics and stability. Research indicates that:
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Tyr272 is located near the lateral interfaces of microtubules, potentially modulating interactions between tubulin dimers .
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Phosphorylation at this site can affect the binding of microtubule-associated proteins (MAPs) that regulate polymerization and depolymerization rates .
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Changes in phosphorylation status at Tyr272 have been associated with microtubule stability, with phosphorylation generally correlated with more dynamic microtubules .
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In neurological contexts, altered phosphorylation at this site has been observed in conditions including Alzheimer's disease, where the phosphorylation subnetwork was found to be highly enriched with differentially expressed genes .
Understanding this modification is particularly relevant in neuronal systems, where precise regulation of microtubule dynamics is essential for neuronal migration, differentiation, and axonal transport .
How should the antibody be stored and handled for optimal performance?
Proper storage and handling are crucial for maintaining antibody activity:
The antibody is typically supplied in phosphate buffered saline (without Mg²⁺ and Ca²⁺), pH 7.4, with 150mM NaCl, 0.02% sodium azide, and 50% glycerol . This formulation helps maintain stability during storage. When handling the antibody, use sterile technique and avoid contamination or exposure to heat or strong light, which can degrade protein structure and compromise function .
How can I verify the phospho-specificity of this antibody in my experimental system?
Validating phospho-specificity is critical for ensuring reliable results. Consider these methodological approaches:
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Phosphatase treatment control: Treat one sample with lambda phosphatase to remove phosphate groups. A true phospho-specific antibody will show reduced or absent signal in the dephosphorylated sample compared to untreated controls.
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Peptide competition assay: Pre-incubate the antibody with:
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Phosphorylated peptide (should block signal)
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Non-phosphorylated peptide (should not affect signal)
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Kinase activation/inhibition: Treat cells with:
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Kinase activators that increase Tyr272 phosphorylation (should increase signal)
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Tyrosine kinase inhibitors (should decrease signal)
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siRNA knockdown of relevant kinases: Identify and knockdown kinases known to phosphorylate tubulin at Tyr272, then confirm decreased signal.
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Ectopic expression studies: Compare wild-type TUBA1A with a Y272F mutant (non-phosphorylatable) to confirm specificity.
Researchers studying tubulin phosphorylation networks have identified connections with HDAC6-related pathways, which may inform experimental design for validation studies . The antibody's purification process, which includes removal of non-phospho-specific antibodies by chromatography, should provide high specificity, but verification in your specific system remains essential .
What is the relationship between tubulin Tyr272 phosphorylation and neurodevelopmental disorders?
Research has established important connections between tubulin phosphorylation and neurodevelopmental conditions:
The detection of phosphorylated tubulins in these contexts can provide insights into how post-translational modifications contribute to neuronal migration, differentiation, and the pathogenesis of neurodevelopmental disorders .
How can I optimize detection of phosphorylated tubulin in different subcellular compartments?
Detecting phosphorylated tubulin in specific subcellular locations requires tailored approaches:
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Subcellular fractionation protocol:
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Separate cytosolic, membrane, and cytoskeletal fractions using differential centrifugation
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For microtubule-specific isolation, use a microtubule stabilization buffer (containing taxol and GTP) prior to fractionation
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Process fractions quickly at 4°C to preserve phosphorylation status
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Immunofluorescence optimization:
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Fixation method: 4% paraformaldehyde preserves phospho-epitopes better than methanol
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Include phosphatase inhibitors (10mM NaF, 1mM Na₃VO₄) in all buffers
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Use detergent permeabilization (0.1% Triton X-100) carefully - excessive permeabilization can extract soluble tubulin
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Co-stain with markers for specific compartments (e.g., Golgi, centrosome, midbody)
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Multi-antibody approach:
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Proximity ligation assay (PLA):
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Use PLA to detect interactions between phosphorylated tubulin and compartment-specific binding partners
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This provides higher specificity for detecting functionally relevant phosphorylated tubulin pools
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Research using alpha-tubulin antibodies has demonstrated successful cytoskeleton staining in cell lines like HeLa, showing distinct localization patterns that can be compared with phospho-specific staining . When analyzing results, consider that tubulin phosphorylation may vary across the microtubule network and during different cell cycle stages.
How does Tyr272 phosphorylation influence microtubule dynamics and stability?
Tyr272 phosphorylation has significant effects on microtubule properties:
To experimentally characterize these effects, researchers can use the Phospho-TUBA1A/TUBA1B/TUBA1C/TUBA3C/TUBA3E/TUBA4A (Tyr272) antibody to quantify phosphorylation levels while simultaneously measuring microtubule dynamics through live-cell imaging of fluorescently tagged tubulin or EB1 (to track growing microtubule plus ends).
How can this antibody be incorporated into advanced single-cell analysis techniques like Phospho-seq?
Integrating Phospho-TUBA1A/TUBA1B/TUBA1C/TUBA3C/TUBA3E/TUBA4A (Tyr272) antibody into single-cell profiling requires specific adaptations:
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Antibody compatibility with Phospho-seq:
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DNA oligo conjugation process:
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The antibody can be conjugated with complex 15 nt indices as part of a larger DNA-oligo
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Use either TSB tags (10X feature barcodes) or TSA tags, with TSB tags being preferred due to potential RBP binding of TSA tags
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For conjugation, use TCO-labeled oligos at approximately 15 pmol oligo per μg of antibody (adjust based on oligo age)
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Multiplexing capabilities:
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Data analysis considerations:
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Analyze correlations between tubulin phosphorylation and expression of specific genes
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Look for cell subpopulations with distinct phosphorylation patterns
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Integrate with other single-cell data to create multi-omic profiles
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When implementing this technique, researchers should first validate the conjugated antibody independently through flow cytometry to ensure that oligo conjugation hasn't impaired binding specificity. The storage of conjugated antibodies at 4°C should maintain functionality for at least a year, though TCO-labeled oligos may gradually lose their TCO-label over time .
What methodological approaches can improve detection of low-abundance phosphorylated tubulin in complex samples?
Detecting low-abundance phosphorylated tubulin presents challenges that can be addressed through specialized techniques:
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Phosphoprotein enrichment strategies:
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Immobilized metal affinity chromatography (IMAC) using Fe³⁺ or Ga³⁺ columns
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Titanium dioxide (TiO₂) enrichment specifically for phosphopeptides
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Phospho-specific immunoprecipitation using the Phospho-TUBA1A/TUBA1B/TUBA1C/TUBA3C/TUBA3E/TUBA4A (Tyr272) antibody
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Signal amplification methods:
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Tyramide signal amplification (TSA) for immunofluorescence
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Enhanced chemiluminescence (ECL) with high-sensitivity substrates for Western blots
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Proximity ligation assay (PLA) to detect specific interactions involving phosphorylated tubulin
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Sample preparation optimization:
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Include phosphatase inhibitors (sodium orthovanadate, sodium fluoride, β-glycerophosphate) in all buffers
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Maintain samples at 4°C during processing
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Use proteasome inhibitors to prevent degradation of phosphorylated proteins
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Consider heat-stabilization methods that rapidly inactivate phosphatases
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Advanced detection platforms:
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Nano-flow liquid chromatography coupled to high-resolution mass spectrometry
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Selected reaction monitoring (SRM) or parallel reaction monitoring (PRM) for targeted detection
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Digital Western blot platforms with increased sensitivity and dynamic range
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For experimental control and validation, parallel analysis of samples treated with tyrosine phosphatase inhibitors or tyrosine kinase activators can provide positive controls with increased signals. Researchers studying phosphorylation networks in neurological disorders have employed such approaches to detect subtle changes in tubulin phosphorylation states .
How can this antibody be used to investigate the relationship between tubulin phosphorylation and HDAC6 activity?
HDAC6 and tubulin phosphorylation exhibit complex interactions that can be studied using this antibody:
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Experimental design to investigate HDAC6-phosphorylation interactions:
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Treat cells with specific HDAC6 inhibitors (tubastatin A, ACY-1215) and measure changes in TUBA1A/TUBA1B/TUBA1C/TUBA3C/TUBA3E/TUBA4A Tyr272 phosphorylation
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Perform HDAC6 knockdown/overexpression and assess effects on tubulin phosphorylation status
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Combine with acetylation-specific tubulin antibodies to examine the relationship between these modifications
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Relevant biological contexts:
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Neurodegeneration models: HDAC6 regulatory mechanisms have been implicated in Alzheimer's disease, showing connections to phosphorylation networks
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Epithelial organization: HDAC6 suppression reverses oncogenic Shp2-induced multiple apical domains and spindle misorientation
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Cancer models: Both HDAC6 activity and tubulin phosphorylation are altered in various cancer types
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Detection protocol for dual modifications:
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Sequential immunoprecipitation (IP) using anti-acetylated tubulin followed by Western blot with Phospho-TUBA1A/TUBA1B/TUBA1C/TUBA3C/TUBA3E/TUBA4A (Tyr272) antibody
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Alternatively, IP with Phospho-TUBA1A/TUBA1B/TUBA1C/TUBA3C/TUBA3E/TUBA4A (Tyr272) antibody followed by probing for acetylation
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Double immunofluorescence staining to visualize co-localization patterns
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Mechanistic investigations:
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Examine if HDAC6-mediated deacetylation affects accessibility of Tyr272 for phosphorylation
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Assess whether phosphorylation at Tyr272 influences HDAC6 binding to tubulin
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Investigate downstream effects on microtubule dynamics and cellular functions
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Research has shown that HDAC6 inhibition causes changes in Cdc42 activity and affects myosin II and ERK1/2 activity, which may indirectly influence tubulin phosphorylation states . Using the Phospho-TUBA1A/TUBA1B/TUBA1C/TUBA3C/TUBA3E/TUBA4A (Tyr272) antibody in combination with phospho-specific antibodies against these signaling molecules can provide a comprehensive view of the signaling networks involved.
