TUBA1A Monoclonal Antibody

What is TUBA1A and why is it significant in research?

TUBA1A is an alpha-tubulin isoform that constitutes the majority of α-tubulin in the developing brain. It forms heterodimers with β-tubulin to create microtubules, essential components of the eukaryotic cytoskeleton that perform diverse cellular functions. TUBA1A is particularly significant in neurodevelopmental research because mutations in the TUBA1A gene cause severe brain malformations, including lissencephaly, polymicrogyria, and other cortical development disorders collectively termed tubulinopathies . The high conservation of this protein across species (100% identity between human and rat homologs, with only 2-3 amino acid differences compared to pig and chicken homologs) makes it an excellent target for studying evolutionary conserved microtubule functions .

How do I select the appropriate TUBA1A monoclonal antibody for my experimental system?

Selection requires careful consideration of multiple factors:

• Species reactivity: Confirm the antibody cross-reacts with your experimental species. For example, clone DM1A reacts with TUBA1A in chicken, human, mouse, and rat samples , while other antibodies may have more limited reactivity profiles.

• Application compatibility: Verify validation for your specific application. Some antibodies are validated for multiple applications with different recommended dilutions:

• Clone specificity: Determine if the antibody recognizes only TUBA1A or also detects other alpha-tubulin isoforms. This is critical as mammalian genomes contain multiple α-tubulin genes encoding highly similar proteins, leading to antibody promiscuity .

• Epitope location: Consider whether the epitope region might be masked in your experimental context (e.g., by protein-protein interactions or post-translational modifications).

What is the expected molecular weight of TUBA1A in Western blot applications?

TUBA1A has a calculated molecular weight of 50,136 Da, but is typically observed at approximately 55 kDa in Western blot applications . This discrepancy between calculated and observed molecular weights is common for many proteins due to post-translational modifications, differences in electrophoretic mobility, or the influence of buffer conditions. When performing Western blot analysis, researchers should include positive controls such as HeLa, HEK293, or Raji cell lysates, which have been validated to express detectable levels of TUBA1A .

What are the optimal dilution conditions for TUBA1A monoclonal antibodies in different applications?

Different applications require specific antibody concentrations for optimal signal-to-noise ratio:

The optimal working dilution should ultimately be determined empirically by each researcher, as factors such as tissue type, fixation method, and detection system can influence antibody performance . For most applications, a titration experiment with 3-5 different concentrations is recommended to determine the optimal signal-to-background ratio.

How should TUBA1A antibodies be stored and handled to maintain reactivity?

Proper storage and handling are critical for maintaining antibody performance:

• Storage temperature:

  • Lyophilized antibodies (e.g., MA1107): Store at -20°C for up to one year from receipt date .

  • Liquid formulations (e.g., A03989): Store at 4°C for up to three months or at -20°C for up to one year .

• Reconstitution protocols:

  • For lyophilized antibodies, add the recommended volume of sterile buffer (typically PBS) to achieve the desired concentration. For example, adding 1 ml of PBS to MA1107 yields a concentration of 100 μg/ml .

  • After reconstitution, store at 4°C for short-term use (up to one month).

• Aliquoting recommendations:

  • For long-term storage, divide the reconstituted antibody into small single-use aliquots to avoid repeated freeze-thaw cycles.

  • Aliquots can typically be stored at -20°C for up to six months .

• Avoiding contaminants:

  • Use sterile technique when handling antibodies.

  • Be aware that some preparations contain sodium azide (0.01-0.03%) as a preservative, which is toxic and should be handled accordingly .

How do I optimize immunofluorescence staining protocols for TUBA1A detection in neuronal tissues?

Detecting TUBA1A in neuronal tissues requires careful optimization:

• Fixation method: For preserved microtubule structure, use 4% paraformaldehyde for 15-20 minutes at room temperature. Avoid methanol fixation which can disrupt microtubule organization.

• Permeabilization: Use 0.1-0.3% Triton X-100 in PBS for 10 minutes to allow antibody access to intracellular antigens.

• Blocking: Incubate with 5-10% normal serum (from the species in which the secondary antibody was raised) with 1% BSA in PBS for 1 hour at room temperature.

• Primary antibody: Apply TUBA1A antibody at the recommended concentration (typically 20 μg/ml for immunofluorescence) . Incubate overnight at 4°C for optimal results.

• Washing: Perform 3-5 washes with PBS containing 0.1% Tween-20 to reduce background.

• Secondary antibody: Use fluorophore-conjugated secondary antibodies at 1:200-1:500 dilutions for 1-2 hours at room temperature. Protect from light.

• Counterstaining: DAPI (1:1000) can be used to visualize nuclei.

• Mounting: Use anti-fade mounting medium to prevent photobleaching.

The validation images from antibody A03989 demonstrate successful immunofluorescence staining of TUBA1A in human brain tissue samples at 20 μg/ml concentration .

How can I validate the specificity of a TUBA1A monoclonal antibody in my experimental system?

Ensuring antibody specificity is crucial, especially given the high sequence similarity among alpha-tubulin isoforms:

• Positive controls: Include cell lines with known TUBA1A expression such as HeLa, HEK293, or Raji cells, which have been validated for TUBA1A detection .

• Negative controls:

  • Omit primary antibody but include all other steps

  • Use isotype control antibodies (e.g., mouse IgG1 for DM1A clone)

  • When possible, use TUBA1A-knockout or knockdown samples

• Cross-reactivity testing: If working with multiple species, confirm the antibody detects bands of the expected molecular weight in each species.

• Peptide competition assay: Pre-incubate the antibody with purified TUBA1A peptide before application to verify that this blocks specific binding.

• Multi-antibody validation: Compare results using two different TUBA1A antibodies targeting distinct epitopes.

• Western blot profile: A clean single band at approximately 55 kDa suggests specificity, whereas multiple bands may indicate cross-reactivity with other tubulin isoforms .

Boster Bio validates all antibodies with known positive control and negative samples using multiple techniques (WB, IHC, ICC, Immunofluorescence, and ELISA) to ensure specificity and high affinity .

What are common technical issues when using TUBA1A antibodies and how can they be resolved?

For Western blot applications specifically, comparing results from HeLa, HEK293, and Raji cell lysates can help identify the optimal conditions for TUBA1A detection, as these have been validated as reliable positive controls .

How can TUBA1A monoclonal antibodies be used to study tubulinopathies and neurodevelopmental disorders?

TUBA1A mutations cause severe brain malformations, and monoclonal antibodies provide valuable tools for investigating these conditions:

• Mutation-specific effects: Researchers can transfect cells with wild-type or mutant TUBA1A and use antibodies to compare protein levels, localization, and incorporation into microtubules. Studies have shown that disease-causing mutations like I188L, I238V, P263T, L286F, V303G, L397P, R402C, R402H, and S419L produce tubulin heterodimers in varying yields and can co-polymerize with microtubules in vitro, but may affect chaperone-dependent pathways and microtubule dynamics .

• Developmental expression patterns: Immunohistochemistry with TUBA1A antibodies can reveal spatiotemporal expression patterns during brain development, helping to understand why mutations cause specific malformations like lissencephaly or polymicrogyria.

• Protein-protein interactions: Immunoprecipitation with TUBA1A antibodies can identify interactions with microtubule-associated proteins (MAPs) and how these might be disrupted by disease-causing mutations.

• Live-cell imaging: Combining TUBA1A antibodies with novel tagging methods allows visualization of microtubule dynamics in developing neurons without impairing tubulin function .

• Functional assays: TUBA1A antibodies can be used to evaluate how mutations affect neurite extension, growth cone composition, and commissural axon architecture during brain development .

How do disease-causing mutations in TUBA1A affect protein folding and heterodimer assembly pathways?

Disease-causing mutations in TUBA1A disrupt key steps in the complex tubulin folding and assembly pathway:

These findings suggest two primary disease mechanisms: tubulin deficit due to compromised heterodimer formation, and disrupted interactions with MAPs essential for proper neuronal migration .

What are the key experimental considerations when using TUBA1A antibodies to study microtubule dynamics in neuronal growth cones?

Studying microtubule dynamics in neuronal growth cones presents unique challenges:

• Fixation methodology: Standard paraformaldehyde fixation may not preserve the dynamic microtubule structures in growth cones. Consider using specialized fixatives such as PHEM buffer (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, 2 mM MgCl₂, pH 6.9) with 0.25% glutaraldehyde to better preserve microtubule ultrastructure.

• Co-visualization strategies: Combine TUBA1A antibody staining with markers for:

  • Actin filaments (phalloidin) to examine microtubule-actin interactions

  • Post-translational modifications (acetylation, tyrosination) to distinguish stable vs. dynamic microtubule populations

  • End-binding proteins (EB1/3) to visualize growing microtubule plus-ends

• Super-resolution imaging: Conventional fluorescence microscopy has limited resolution for the fine architecture of growth cone microtubules. Consider:

  • Structured illumination microscopy (SIM)

  • Stimulated emission depletion (STED) microscopy

  • Stochastic optical reconstruction microscopy (STORM)

• Live imaging optimization: For live studies of TUBA1A dynamics:

  • Use novel tagging methods that don't disrupt tubulin function

  • Consider photobleaching techniques (FRAP) to measure microtubule turnover rates

  • Optimize imaging intervals and duration to minimize phototoxicity

• Quantification approaches: Develop robust methods to quantify:

  • Microtubule growth rates (typically 5-15 μm/min in growth cones)

  • Catastrophe and rescue frequencies

  • Microtubule looping and bundling

  • Invasion of microtubules into the peripheral domain

• Pharmacological manipulations: Include controls with microtubule-disrupting (nocodazole) or -stabilizing (taxol) agents at low concentrations to validate the specificity of observed dynamics.

Research has shown that reduced levels of Tuba1a affect commissure formation but can still support neuronal migration and cortex development, highlighting the importance of studying dosage effects on specific neuronal processes .

How can novel tagging methods improve the study of TUBA1A in developing neurons?

Traditional tagging approaches for tubulin often disrupt its function, but recent methodologies offer promising alternatives:

• Genetic tagging approaches: Researchers have developed novel tagging methods for studying and manipulating TUBA1A in cells without impairing tubulin function. These techniques allow visualization of TUBA1A dynamics during neuronal development and can be used to investigate how mutations affect microtubule behavior in living cells .

• Advantages over antibody-based detection:

  • Enables live-cell imaging of TUBA1A dynamics

  • Allows distinction between TUBA1A and other alpha-tubulin isoforms

  • Permits tracking of newly synthesized vs. existing tubulin pools

  • Facilitates study of tubulin post-translational modifications in situ

• Implementation considerations:

  • Tag placement must avoid interference with tubulin folding, heterodimer formation, or MAP binding

  • Expression levels should be carefully controlled to prevent artifacts

  • Validation against antibody-based detection is essential to confirm normal function

• Applications to disease models: These tagging methods can be combined with disease-causing mutations to directly visualize how mutations alter TUBA1A behavior in developing neurons, particularly in processes like neurite extension and growth cone dynamics .

What approaches can differentiate between the roles of TUBA1A and other alpha-tubulin isoforms in experimental systems?

Distinguishing the specific roles of TUBA1A from other alpha-tubulin isoforms presents significant challenges due to their high sequence similarity:

• Isoform-specific knockdown/knockout: Use siRNA or CRISPR-Cas9 targeting the unique 3'-UTR of TUBA1A, which is more than 80% homologous to the UTR of the rat brain alpha-tubulin gene IL-alpha-T1 but differs from other isoforms .

• Rescue experiments: After knockdown of endogenous alpha-tubulins, express TUBA1A or other isoforms to identify specific functions. This approach has shown that TUBA1A constitutes the majority of α-tubulin in the developing brain and has critical roles in neuronal migration, cortex development, and commissure formation .

• Mass spectrometry-based approaches:

  • Targeted mass spectrometry can distinguish between alpha-tubulin isoforms based on isoform-specific peptides

  • Quantitative proteomics can measure the relative abundance of different isoforms across tissues and developmental stages

• Developmental expression analysis: Combine RNA-Seq with isoform-specific antibodies to track developmental regulation of TUBA1A compared to other alpha-tubulin genes.

• Isoform-specific post-translational modifications: Investigate whether certain modifications preferentially occur on TUBA1A compared to other isoforms, potentially linking to functional specificity.

How can TUBA1A antibodies be used to investigate the relationship between microtubule dynamics and neuronal migration defects?

Neuronal migration defects are a hallmark of TUBA1A-related tubulinopathies, and antibodies can help elucidate the underlying mechanisms:

• Cortical development models:

  • In utero electroporation with wild-type or mutant TUBA1A followed by immunohistochemistry to track migrating neurons

  • Time-lapse imaging of brain slices with fluorescently labeled TUBA1A to visualize migration in real-time

  • Comparison of microtubule organization in normally migrating versus arrested neurons

• Centrosome-microtubule coupling analysis:

  • Co-immunostaining of TUBA1A with centrosomal markers (γ-tubulin, pericentrin)

  • Quantification of centrosome-nucleus distance during migration

  • Assessment of microtubule nucleation from the centrosome in normal vs. mutant conditions

• Cytoskeletal coupling studies:

  • Analysis of interactions between microtubules and actin during leading process extension

  • Evaluation of adhesion molecule distribution in migrating neurons with TUBA1A mutations

  • Quantification of microtubule stability in different cellular compartments during migration

• Growth cone dynamics:

  • Detailed analysis of microtubule behavior in the growth cones of neurons expressing wild-type vs. mutant TUBA1A

  • Investigation of how altered microtubule dynamics affect growth cone navigation and pathfinding

  • Correlation between microtubule growth rates and neurite extension capabilities

• Molecular motor interactions:

  • Assessment of how TUBA1A mutations affect binding and processivity of molecular motors like kinesin and dynein

  • Investigation of cargo transport deficits in neurons with TUBA1A mutations

  • Analysis of nucleokinesis mechanisms dependent on intact microtubule networks

Research has demonstrated that while reduced TUBA1A levels can support neuronal migration and cortex development, they are insufficient for proper commissure formation, suggesting differential sensitivity of specific neurodevelopmental processes to TUBA1A function .