TUBA1A Monoclonal Antibody

What is TUBA1A and why is it important in neuroscience research?

TUBA1A is the most highly expressed α-tubulin isotype in post-mitotic neurons during brain development. It serves as a major constituent of microtubules, which are essential for neuronal migration, axon formation, and dendritic arborization. TUBA1A plays crucial roles in cell division, intracellular transport, and maintenance of cell morphology. Its significance in neuroscience stems from the association between TUBA1A mutations and various neurodevelopmental disorders, including microcephaly, lissencephaly, and intellectual disability . Research using TUBA1A antibodies helps elucidate mechanisms of normal brain development and pathological conditions arising from tubulin dysfunction.

What criteria should be considered when selecting a TUBA1A monoclonal antibody?

When selecting a TUBA1A monoclonal antibody, researchers should consider:

• Epitope specificity: Verify which region of TUBA1A the antibody recognizes (e.g., DM1A clone recognizes an epitope in microtubules from chicken embryo brain)

• Cross-reactivity: Determine if the antibody cross-reacts with other tubulin isotypes or species of interest (e.g., antibody MA1107 reacts with chicken, human, mouse, and rat TUBA1A)

• Validated applications: Confirm the antibody has been validated for your specific application (WB, IHC, IF, IP, FC)

• Clone information: Consider established clones with publication history (e.g., DM1A clone for TUBA1A)

• Formulation compatibility: Ensure the antibody formulation is compatible with your experimental conditions

Consider comparing antibody performance data from vendor validation studies to select the optimal reagent for your specific research application.

How do I properly reconstitute and store TUBA1A monoclonal antibodies to maintain activity?

TUBA1A monoclonal antibodies require careful handling to maintain functionality:

Reconstitution protocol:

• Allow the lyophilized antibody to reach room temperature before opening

• For lyophilized antibodies (e.g., MA1107), add 1ml of PBS buffer to yield a concentration of 100μg/ml

• Gently mix by pipetting or rotating; avoid vigorous vortexing which can damage antibody structure

Storage recommendations:

• Store unopened lyophilized antibody at -20°C for up to one year from date of receipt

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

• For long-term storage, aliquot and freeze at -20°C for up to six months

• Avoid repeated freeze-thaw cycles which significantly reduce antibody activity

Working solution preparation:

• Prepare fresh working dilutions on the day of the experiment

• Some antibodies (e.g., CSB-MA754656A0m) are supplied in a storage buffer containing PBS, pH 7.4, with 0.02% sodium azide as preservative and 50% glycerol

What are the optimal dilution ranges for TUBA1A antibodies across different applications?

Optimal dilution ranges vary by antibody clone and application:

Always perform a dilution series optimization experiment for your specific sample type and detection system. The observed molecular weight of TUBA1A is approximately 55 kDa, with a calculated molecular weight of 50.1 kDa .

How can I validate the specificity of a TUBA1A monoclonal antibody?

To validate TUBA1A antibody specificity:

• Positive controls: Use tissues/cells known to express high levels of TUBA1A (e.g., brain tissue, particularly developing neurons)

• Molecular weight verification: Confirm a single band at approximately 55 kDa in Western blot analysis

• Cross-reactivity assessment: Test the antibody against multiple species if doing comparative studies (e.g., CSB-MA754656A0m reacts with human, rabbit, rat, and mouse TUBA1A)

• Knockdown/knockout validation: Compare antibody signal in TUBA1A knockdown/knockout samples versus wild-type controls

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide prior to application; specific binding should be blocked

• Multiple antibody comparison: Use two antibodies recognizing different epitopes of TUBA1A and compare staining patterns

• Immunoprecipitation followed by mass spectrometry: Verify that TUBA1A is the predominant protein pulled down

What control samples should I include when using TUBA1A antibodies in neuronal research?

For rigorous neuronal research with TUBA1A antibodies, include:

Positive controls:

• Brain tissue samples (particularly cerebral cortex) where TUBA1A is highly expressed

• Neuronal cell lines (e.g., primary neuronal cultures or neuroblastoma lines)

• HeLa cells, which have been validated for some TUBA1A antibodies

Negative controls:

• Secondary antibody-only controls to assess background

• Non-neuronal tissue with minimal TUBA1A expression

• For mutation studies, wild-type samples compared to TUBA1A mutants

Experimental controls:

• When studying TUBA1A mutations, include both wild-type and mutant samples (e.g., V409I and V409A mutants show different severity scales)

• Include multiple developmental timepoints if studying neurogenesis

• For microtubule dynamics studies, include samples treated with stabilizing (taxol) or destabilizing (nocodazole) agents

How can TUBA1A antibodies be used to investigate tubulinopathy-associated mutations?

TUBA1A antibodies are valuable tools for investigating tubulinopathy-associated mutations:

• Mutation-specific phenotypic analysis: Compare wild-type TUBA1A localization and expression with mutant forms (e.g., V409I/A) to correlate with severity of neural phenotypes

• Microtubule dynamics assessment: Use real-time immunofluorescence to measure differences in microtubule polymerization and depolymerization rates between wild-type and mutant TUBA1A, as demonstrated with V409I/A mutants that showed increased polymerization rates

• Post-translational modification profiling: Combine TUBA1A antibodies with PTM-specific antibodies (e.g., acetylation) to determine how mutations affect modifications that regulate microtubule stability

• Interaction partner identification: Use co-immunoprecipitation with TUBA1A antibodies to identify how mutations alter binding to regulatory proteins like XMAP215/Stu2

• In vivo migration studies: Apply TUBA1A antibodies in fixed tissue sections to evaluate neuronal migration defects in animal models expressing TUBA1A mutations

Methodologically, researchers should include antibodies against other microtubule components and regulatory proteins to build a comprehensive understanding of how specific mutations disrupt normal microtubule function and neuronal development.

What methods can be used to study TUBA1A interactions with microtubule-associated proteins?

To investigate TUBA1A interactions with microtubule-associated proteins (MAPs):

• Co-immunoprecipitation (Co-IP): Use TUBA1A antibodies to pull down protein complexes, followed by Western blot analysis for specific MAPs or mass spectrometry for unbiased discovery of interaction partners

• Proximity ligation assay (PLA): Detect in situ interactions between TUBA1A and MAPs with spatial resolution at the subcellular level

• FRET/FLIM analysis: Measure direct protein-protein interactions and their dynamics in living cells

• In vitro reconstitution assays: Combine purified TUBA1A and MAPs to study direct interactions and functional effects on microtubule dynamics

• Yeast two-hybrid screening: Identify novel TUBA1A-interacting proteins

• Crosslinking mass spectrometry: Map interaction interfaces between TUBA1A and MAPs at amino acid resolution

• Super-resolution microscopy: Visualize co-localization of TUBA1A and MAPs with nanometer precision

For example, when studying the relationship between TUBA1A mutations and XMAP215/Stu2 recruitment, combine co-immunoprecipitation with in vivo imaging to correlate biochemical interactions with functional outcomes in microtubule polymerization dynamics .

How can I use TUBA1A antibodies to investigate microtubule dynamics in neural development and disease models?

To investigate microtubule dynamics in neural development and disease models using TUBA1A antibodies:

• Live-cell imaging with fluorescently tagged TUBA1A antibody fragments: Monitor microtubule dynamics in real-time in developing neurons or disease models

• Pulse-chase experiments: Use TUBA1A antibodies to track newly synthesized tubulin incorporation into microtubules during neuronal development

• Correlative light-electron microscopy: Combine TUBA1A immunofluorescence with ultrastructural analysis to link microtubule organization to cellular morphology

• Multi-parameter analysis in disease models: Compare TUBA1A distribution, post-translational modifications, and binding partners between normal and pathological states

• Organoid models: Apply TUBA1A antibodies in cerebral organoids to study neurodevelopmental disorders in a 3D context

Specifically, TUBA1A antibodies have revealed that V409I/A mutations promote excessive neurite branching and decrease neurite retraction in primary rat neuronal cultures, accompanied by increased microtubule acetylation and polymerization rates . This demonstrates how TUBA1A antibodies can help establish mechanistic links between molecular alterations and cellular phenotypes in disease models.

What are common issues when using TUBA1A antibodies in Western blot analysis and how can they be resolved?

For optimization:

• When working with brain tissue samples, ensure proper homogenization and add protease inhibitors to prevent degradation

• For dilution optimization, start with mid-range dilution (e.g., 1:5000 for MACO0009) and adjust based on signal intensity

• Consider the species reactivity of your antibody (e.g., MA1107 reacts with chicken, human, mouse, rat)

How can I distinguish between TUBA1A and other alpha-tubulin isotypes in my experiments?

Distinguishing TUBA1A from other alpha-tubulin isotypes requires careful experimental design:

• Epitope-specific antibody selection: Choose antibodies that target unique regions of TUBA1A not conserved in other isotypes. Monoclonal antibodies like DM1A have been extensively validated for TUBA1A specificity

• Expression pattern analysis: TUBA1A is predominantly expressed in post-mitotic neurons in the developing brain, which can help distinguish it from other isotypes in tissue-specific contexts

• mRNA detection methods: Combine protein detection with mRNA analysis (RT-PCR, RNA-Seq, or in situ hybridization) 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

• Genetic manipulation: Use TUBA1A-specific knockdown or overexpression systems as controls

• Mass spectrometry validation: Confirm antibody specificity by immunoprecipitation followed by mass spectrometry to identify the exact tubulin isotype being detected

• Cross-reactivity testing: Test antibody against recombinant proteins of different tubulin isotypes to establish specificity profiles

What considerations are important when interpreting TUBA1A immunostaining patterns in neuronal tissues?

When interpreting TUBA1A immunostaining in neuronal tissues:

• Developmental timing: TUBA1A expression changes during development, with highest expression in post-mitotic neurons in the developing brain . Interpret staining intensity in the context of developmental stage.

• Subcellular localization: Normal TUBA1A distribution should follow microtubule organization in neurons. In healthy neurons, expect:

  • Uniform distribution throughout the cell body

  • Linear patterns along axons

  • More complex branching patterns in dendrites

• Mutation-specific patterns: TUBA1A mutations can alter subcellular distribution. For example, V409I/A mutations promote excessive neurite branching and affect microtubule acetylation .

• Co-localization analysis: Combine TUBA1A staining with markers for specific neuronal compartments (MAP2 for dendrites, Tau for axons) or post-translational modifications (acetylation, tyrosination).

• Resolution limitations: Standard immunofluorescence cannot resolve individual microtubules; super-resolution techniques may be needed for detailed analysis.

• Fixation artifacts: Different fixation methods affect microtubule preservation and antibody accessibility. Compare multiple fixation protocols to confirm patterns.

• Background discrimination: TUBA1A is abundant, so distinguish specific signal from background by including appropriate controls.

How can TUBA1A antibodies be utilized in studying the relationship between microtubule dynamics and neurodevelopmental disorders?

TUBA1A antibodies provide valuable tools for investigating the mechanistic connections between microtubule dynamics and neurodevelopmental disorders:

• Mutation phenotyping: Compare microtubule organization and dynamics in cells expressing wild-type versus mutant TUBA1A (e.g., V409I/A) to establish genotype-phenotype correlations. For example, V409I/A mutants showed increased microtubule polymerization rates that scaled with mutation severity .

• Combined immunohistochemistry approaches: Use TUBA1A antibodies alongside markers for neuronal migration, axon guidance, and synaptogenesis to comprehensively assess how tubulin mutations affect multiple aspects of neurodevelopment.

• Patient-derived models: Apply TUBA1A antibodies in induced pluripotent stem cell (iPSC)-derived neurons from patients with TUBA1A mutations to study disease-relevant phenotypes in human cells.

• In vivo developmental analysis: Use TUBA1A antibodies to track neuronal migration patterns in animal models expressing tubulinopathy-associated mutations.

• Post-translational modification profiling: Investigate how disease-causing mutations affect tubulin post-translational modifications using specific antibodies for acetylation, tyrosination, and other modifications.

Research has demonstrated that TUBA1A-V409I/A mutations disrupt neuronal migration in mice and promote excessive neurite branching with decreased neurite retraction in primary neuronal cultures . These findings illustrate how TUBA1A antibodies help establish direct links between molecular alterations and cellular phenotypes in neurodevelopmental disorders.

What methods can be used to study TUBA1A post-translational modifications and their impact on microtubule functions?

To investigate TUBA1A post-translational modifications (PTMs) and their functional impacts:

• PTM-specific antibodies: Combine general TUBA1A antibodies with modification-specific antibodies (acetylation, tyrosination, phosphorylation, polyglutamylation) to detect specific modified forms.

• Sequential immunoprecipitation: First immunoprecipitate total TUBA1A, then probe with PTM-specific antibodies to quantify modification levels.

• Mass spectrometry analysis: Use immunoprecipitation with TUBA1A antibodies followed by mass spectrometry to comprehensively profile PTM patterns and identify novel modifications.

• Site-specific mutant comparisons: Compare PTM patterns between wild-type TUBA1A and disease-associated mutants to identify alterations in modification profiles.

• Enzyme inhibitor studies: Treat samples with deacetylase inhibitors or other enzyme modulators to assess how dynamic regulation of PTMs affects TUBA1A function.

• Live-cell imaging: Combine TUBA1A antibody fragments with PTM sensors to monitor modification dynamics in real time.

Research has shown that TUBA1A V409I/A mutations are associated with increased microtubule acetylation , demonstrating how mutations can affect PTM profiles and subsequently alter microtubule dynamics and neuronal development.

How can advanced imaging techniques be combined with TUBA1A antibodies to investigate microtubule dynamics?

Combining advanced imaging techniques with TUBA1A antibodies enables sophisticated analysis of microtubule dynamics:

• Super-resolution microscopy (STORM, PALM, SIM): Overcome the diffraction limit to resolve individual microtubules labeled with TUBA1A antibodies, allowing detailed analysis of microtubule organization and density beyond conventional microscopy capabilities.

• Live-cell single-molecule tracking: Use fluorescently labeled TUBA1A antibody fragments to track incorporation of tubulin dimers into growing microtubules with nanometer precision.

• FRAP (Fluorescence Recovery After Photobleaching): Measure microtubule turnover rates by photobleaching TUBA1A antibody-labeled regions and monitoring fluorescence recovery.

• Correlative light-electron microscopy (CLEM): Combine TUBA1A immunofluorescence with electron microscopy to correlate light microscopy observations with ultrastructural details of microtubule organization.

• Expansion microscopy: Physically expand samples labeled with TUBA1A antibodies to achieve super-resolution imaging on standard microscopes.

• Lattice light-sheet microscopy: Achieve high-speed, low-phototoxicity imaging of TUBA1A dynamics in living neurons over extended periods.

• Fluorescence fluctuation spectroscopy: Measure TUBA1A diffusion rates and incorporation into microtubules.

In studies of TUBA1A mutations, these techniques have revealed that V409I/A mutants promote intrinsically faster microtubule polymerization rates in cells and in reconstitution experiments with purified tubulin , demonstrating the power of combining advanced imaging with specific antibodies to uncover molecular mechanisms of disease.