TUBB2A Monoclonal Antibody

What is TUBB2A and why is it significant in cellular research?

TUBB2A (Tubulin Beta-2A Chain) is a member of the tubulin protein family that plays a crucial role in microtubule formation and dynamics. It contributes to essential cellular processes including mitosis, cell migration, and neuronal development. TUBB2A forms heterodimers with alpha-tubulin that polymerize to create microtubules, cylindrical structures composed of laterally associated linear protofilaments . These microtubules grow by the addition of GTP-tubulin dimers at their ends, where a stabilizing cap forms. The importance of TUBB2A extends beyond basic structural roles, as dysregulation has been linked to neurodevelopmental disorders and cancer progression, making it both a valuable research target and potential therapeutic focus .

What experimental applications are suitable for TUBB2A monoclonal antibodies?

TUBB2A monoclonal antibodies are versatile tools applicable to multiple experimental techniques. Based on validated applications, researchers can confidently employ these antibodies in:

The choice of application should be guided by your specific research questions, with consideration given to the nature of your samples and the level of TUBB2A expression in your experimental system .

How should TUBB2A antibodies be validated for experimental use?

Proper validation of TUBB2A antibodies is essential for research reliability. A comprehensive validation approach should include:

• Positive and negative controls: Use cell lines with known TUBB2A expression levels, such as Jurkat, NIH/3T3, PC-12, or Raw 264.7 cells as positive controls . For negative controls, consider using non-transfected cells alongside TUBB2A-transfected cells .

• Specificity testing: Perform Western blot analysis to confirm the antibody detects a band of the expected molecular weight (~49.9 kDa for TUBB2A) . Cross-reactivity should be assessed, particularly with other beta-tubulin isoforms.

• Application-specific validation: For immunohistochemistry, verify specificity using appropriate tissue sections (e.g., human spleen has been validated) . For immunofluorescence, test on well-characterized cell lines like HeLa .

• Knockdown/knockout verification: Where possible, validate antibody specificity using TUBB2A knockdown or knockout samples to confirm signal reduction or elimination .

• Compare multiple antibody clones: Different clones may perform better in specific applications, so testing multiple antibodies can identify the optimal reagent for your research needs .

What cell types and tissues are recommended for studying TUBB2A expression?

TUBB2A expression has been successfully detected in various cellular and tissue models, with certain systems providing particularly robust results:

When selecting a model system, consider both the endogenous expression level of TUBB2A and the compatibility with your research questions, particularly when studying neurodevelopmental processes or cancer progression .

How can TUBB2A monoclonal antibodies be used to investigate neurodevelopmental disorders?

TUBB2A mutations have been implicated in neurodevelopmental disorders, particularly those characterized by simplified gyral patterning and infantile-onset epilepsy . When designing experiments to investigate these conditions:

• Mutation-specific approaches: TUBB2A mutations affect a highly conserved loop that associates with the alpha-tubulin-bound GTP molecule, impairing the intradimer interface and correct alpha/beta tubulin dimer formation . Design experiments that specifically examine these functional domains.

• Comparative immunostaining: Use TUBB2A antibodies in combination with markers for neuronal migration and cortical organization to assess differences between normal and pathological samples. This approach can reveal how TUBB2A mutations affect neuronal positioning and morphology.

• In vitro modeling: Employ TUBB2A antibodies to study the effects of disease-associated mutations on microtubule dynamics in neuronal cell cultures. This can be achieved by:

  • Comparing wild-type and mutant TUBB2A expression patterns

  • Assessing co-localization with other microtubule components

  • Measuring effects on neuronal migration and neurite extension

• Live-cell imaging: Combine TUBB2A immunofluorescence with time-lapse microscopy to analyze differences in microtubule stability and dynamics between normal and disease models .

• Protein interaction studies: Use immunoprecipitation with TUBB2A antibodies to identify altered protein interactions in disease states, which may reveal pathological mechanisms beyond structural microtubule defects .

What methodological considerations are important when optimizing Western blot protocols for TUBB2A detection?

Western blot optimization for TUBB2A requires attention to several critical factors:

• Sample preparation: TUBB2A is abundant in most cell types but can be sensitive to degradation. Use fresh samples and include protease inhibitors in lysis buffers. For brain tissue, which shows high TUBB2A expression, rapid post-mortem processing is essential to prevent protein degradation .

• Gel percentage selection: TUBB2A has a molecular weight of approximately 49.9 kDa. A 10% SDS-PAGE gel provides optimal resolution for this size range, as demonstrated in validated protocols .

• Transfer conditions: Transfer efficiency can be critical for TUBB2A detection. Use wet transfer methods with methanol-containing buffers for optimal results with tubulin proteins.

• Blocking optimization: 5% non-fat dry milk in TBS-T is typically effective, but for some applications, BSA-based blocking solutions may provide lower background.

• Antibody dilution optimization: Test a range of dilutions to determine optimal signal-to-noise ratio. For TUBB2A antibodies, effective dilutions have been established as 1:500-1:5000 for Western blot applications , with 1:3000 to 1:5000 showing good results in NIH-3T3 and mouse brain lysates .

• Controls:

  • Positive control: Include lysates from cells known to express TUBB2A (Jurkat, NIH/3T3, PC-12, Raw 264.7)

  • Loading control: Use housekeeping proteins other than tubulins to avoid potential cross-reactivity

  • Specificity control: Compare transfected vs. non-transfected lysates when possible

• Signal detection: Both chemiluminescence and fluorescence-based detection systems are suitable, with the latter offering better quantification capabilities for comparative studies.

What are the critical parameters for successful immunofluorescence using TUBB2A antibodies?

Immunofluorescence with TUBB2A antibodies requires careful attention to fixation, permeabilization, and antibody incubation conditions:

• Fixation method selection:

  • Paraformaldehyde (4%) is generally effective for preserving TUBB2A epitopes while maintaining cellular morphology

  • Methanol fixation (cold, -20°C) can enhance microtubule preservation and antibody accessibility

  • Recent studies have compared fixation techniques for cytoskeletal proteins, finding that the optimal method may vary by experimental context and tissue type

• Permeabilization considerations:

  • For PFA-fixed samples, 0.1-0.2% Triton X-100 typically provides good accessibility to microtubule structures

  • Excessive permeabilization can disrupt microtubule architecture, so optimization is essential

• Antibody concentration: 10 μg/ml has been validated for TUBB2A detection in HeLa cells , but optimal concentration should be determined empirically for each cell type

• Blocking parameters: BSA (3-5%) with normal serum from the secondary antibody host species reduces background without interfering with specific binding

• Co-staining considerations:

  • When performing co-localization studies with other microtubule components, careful selection of compatible antibodies from different host species is necessary

  • Sequential staining protocols may be required when using multiple mouse monoclonal antibodies

• Mounting medium selection: Anti-fade mounting media containing DAPI provide nuclear counterstaining while preserving fluorescence signal over time

• Microscopy techniques: Super-resolution techniques (STED, SIM, STORM) can reveal detailed TUBB2A organization within microtubule structures beyond the capabilities of conventional confocal microscopy

How can TUBB2A antibodies be employed in studying interactions with therapeutic compounds targeting microtubules?

TUBB2A is a target for various therapeutic compounds affecting microtubule dynamics. Using TUBB2A antibodies in drug research requires specialized approaches:

• Competitive binding assays: Determine whether drugs directly compete with TUBB2A antibody binding, indicating shared binding sites. This can be assessed through:

  • Pre-incubation of cells/lysates with the compound before antibody application

  • Dose-dependent changes in antibody binding patterns

  • Alterations in epitope accessibility following drug treatment

• Conformation-specific detection: Some TUBB2A antibodies may preferentially recognize specific conformational states of tubulin. These can be leveraged to detect drug-induced conformational changes by:

  • Comparing staining patterns before and after drug treatment

  • Quantifying changes in antibody binding affinity

  • Assessing co-localization with conformation-specific markers

• Post-translational modification monitoring: Many microtubule-targeting agents affect tubulin PTMs. Combined use of TUBB2A antibodies with PTM-specific antibodies can reveal mechanistic insights through:

  • Sequential or multiplexed immunofluorescence

  • Western blot analysis of PTM changes after drug treatment

  • Correlation of PTM patterns with microtubule stability measurements

• Live-cell experimental design: For studying dynamic effects of compounds:

  • Use fluorescently tagged TUBB2A constructs alongside fixed-timepoint antibody staining

  • Employ microfluidic systems for real-time drug addition during imaging

  • Quantify microtubule recovery after drug washout using timed fixation and immunostaining

• Molecular interaction analysis: For compounds suspected to affect TUBB2A protein interactions:

  • Perform immunoprecipitation with TUBB2A antibodies before and after drug treatment

  • Analyze changes in the composition of precipitated protein complexes

  • Identify altered binding partners that may contribute to drug mechanisms or resistance

What considerations are important when studying TUBB2A mutations and their effects on microtubule function?

TUBB2A mutations, particularly de novo mutations associated with simplified gyral patterning and infantile-onset epilepsy, present unique research challenges that can be addressed using specialized approaches with TUBB2A antibodies :

• Epitope accessibility assessment: Mutations may alter antibody binding efficacy. Researchers should:

  • Test multiple antibodies targeting different TUBB2A epitopes

  • Compare staining patterns between wild-type and mutant samples

  • Quantify potential differences in antibody affinity for mutant proteins

• Functional impact analysis: Since mutations in the conserved loop of TUBB2A can impair the intradimer interface and alpha/beta tubulin dimer formation , experiments should be designed to:

  • Compare microtubule polymerization rates in wild-type versus mutant conditions

  • Measure GTP binding and hydrolysis efficiency

  • Assess structural differences in microtubule assemblies

• Cellular phenotype characterization: Using immunofluorescence with TUBB2A antibodies to:

  • Quantify differences in microtubule density and organization

  • Measure alterations in neuronal morphology and migration

  • Analyze cell division abnormalities potentially linked to pathology

• Interaction partner identification: Immunoprecipitation with TUBB2A antibodies can reveal:

  • Changes in binding affinities with other tubulins or microtubule-associated proteins

  • Altered interactions with molecular motors or signaling proteins

  • Potential compensatory mechanisms in mutant cells

• In silico integration: Combining antibody-based experimental data with predictive modeling to:

  • Map mutation locations relative to antibody binding sites

  • Predict structural consequences of mutations

  • Design targeted therapeutic approaches based on specific molecular defects

How can researchers address common issues with TUBB2A antibody specificity?

Despite high specificity of commercial TUBB2A monoclonal antibodies, cross-reactivity issues may arise due to the high homology between tubulin isoforms. To address specificity concerns:

• Validate using molecular weight verification: TUBB2A has a molecular weight of approximately 49.9 kDa . Ensure your Western blot detects a single band at this size.

• Employ knockout/knockdown controls: Compare antibody staining between:

  • Wild-type samples and TUBB2A-depleted samples

  • Transfected and non-transfected cell lines

  • Tissues with known differential expression

• Perform peptide competition assays: Pre-incubate the antibody with the immunizing peptide before application to samples. Specific signals should be abolished or significantly reduced.

• Cross-validate with multiple antibodies: Use antibodies raised against different TUBB2A epitopes and compare staining patterns. Consistent results suggest greater specificity.

• Verify across multiple applications: If an antibody shows consistent results across Western blot, immunofluorescence, and immunohistochemistry, specificity is more likely.

• Consider application-specific optimizations: For instance, in immunohistochemistry, titrate antibody concentration carefully, as the validated concentration of 1.5 μg/ml for FFPE human spleen samples may need adjustment for other tissues .

What controls are essential when using TUBB2A antibodies in experimental workflows?

Proper experimental controls are critical for generating reliable data with TUBB2A antibodies:

• Positive tissue/cell controls:

  • For Western blot: NIH/3T3, Jurkat, PC-12, or Raw 264.7 cell lysates

  • For immunohistochemistry: Human spleen sections

  • For immunofluorescence: HeLa cells

• Negative controls:

  • Primary antibody omission control

  • Isotype control (IgG2a Kappa for many TUBB2A monoclonal antibodies)

  • Non-transfected cell lysates when using overexpression systems

• Loading controls for Western blot:

  • Avoid other tubulins as loading controls due to potential cross-reactivity

  • Use structurally unrelated housekeeping proteins like GAPDH or actin

• Antibody validation controls:

  • Dilution series to establish optimal concentration

  • Batch-to-batch consistency verification

  • Storage time effect assessment (antibody performance over time)

• Technical controls:

  • Multiple biological replicates

  • Randomization of sample processing order

  • Blinded analysis of results when possible

How should researchers interpret contradictory results when using different TUBB2A antibody clones?

When different TUBB2A antibody clones yield contradictory results, systematic analysis is required:

How can TUBB2A antibodies contribute to research on neurodegenerative diseases?

While TUBB2A mutations are directly linked to certain neurodevelopmental disorders , its role in neurodegenerative diseases is an emerging area of investigation. TUBB2A antibodies can facilitate this research through:

• Pathological aggregate analysis: Using immunohistochemistry to:

  • Detect TUBB2A in protein aggregates characteristic of neurodegenerative diseases

  • Analyze co-localization with disease-specific proteins (tau, alpha-synuclein, etc.)

  • Compare TUBB2A distribution in healthy versus diseased tissues

• Microtubule stability assessment: Employing TUBB2A antibodies to:

  • Quantify potential changes in microtubule density in disease states

  • Detect alterations in post-translational modifications affecting stability

  • Measure association with stabilizing or destabilizing factors

• Axonal transport studies: Investigating how TUBB2A abnormalities might contribute to transport deficits by:

  • Analyzing TUBB2A distribution along axons in disease models

  • Assessing co-localization with motor proteins and cargo

  • Measuring dynamic properties of TUBB2A-containing microtubules

• Therapeutic target identification: Using TUBB2A antibodies to:

  • Screen compounds that restore normal TUBB2A function or distribution

  • Identify proteins that interact with TUBB2A in disease-specific contexts

  • Validate TUBB2A-directed therapeutic approaches

What methodological approaches are recommended for studying TUBB2A in cancer progression?

TUBB2A dysregulation has been linked to cancer progression . Researchers investigating this connection can employ TUBB2A antibodies in specialized approaches:

• Expression profiling across cancer types:

  • Use immunohistochemistry with TUBB2A antibodies on tissue microarrays

  • Compare expression levels between normal and malignant tissues

  • Correlate expression with clinical outcomes and treatment responses

• Resistance mechanism investigation:

  • Many cancer therapies target microtubule dynamics

  • TUBB2A antibodies can help identify alterations associated with drug resistance

  • Compare pre- and post-treatment samples for changes in TUBB2A expression or modification

• Migration and invasion analysis:

  • Employ immunofluorescence to track TUBB2A distribution during cancer cell migration

  • Analyze changes in microtubule organization at invasion fronts

  • Correlate TUBB2A patterns with metastatic potential

• Therapeutic response prediction:

  • Develop TUBB2A-based immunohistochemical assays to predict response to microtubule-targeting drugs

  • Identify TUBB2A modification patterns associated with sensitivity or resistance

  • Create combination therapy approaches based on TUBB2A status

• Experimental design considerations:

  • Include multiple cancer cell lines with varying TUBB2A expression levels

  • Validate antibody specificity in each cell line before proceeding

  • Consider three-dimensional culture systems to better approximate in vivo conditions

How can researchers effectively use TUBB2A antibodies in multiplex imaging studies?

Advanced multiplex imaging with TUBB2A antibodies allows simultaneous visualization of multiple markers, providing rich contextual information:

• Antibody selection for multiplexing:

  • Choose TUBB2A antibodies from different host species than other target antibodies

  • Verify that antibodies maintain specificity under multiplexing conditions

  • Test for potential cross-reactivity between detection systems

• Sequential multiplexing approaches:

  • When using antibodies from the same host species, consider sequential staining with stripping between rounds

  • Validate that epitope detection is not compromised by previous staining/stripping cycles

  • Document potential signal reduction through repeated cycles

• Spectral unmixing considerations:

  • Select fluorophores with minimal spectral overlap

  • Include single-stain controls for accurate unmixing

  • Consider using quantum dots or other narrow-emission fluorophores for crowded panels

• Sample preparation optimization:

  • Microtubule preservation is critical for TUBB2A studies

  • Test multiple fixation protocols to identify optimal conditions for simultaneous preservation of all targets

  • Consider specialized fixatives developed for cytoskeletal proteins

• Analysis strategy development:

  • Design quantification approaches for co-localization measurement

  • Establish thresholds for positive staining based on controls

  • Consider machine learning approaches for pattern recognition in complex datasets

What are the optimal storage conditions for maximizing TUBB2A antibody shelf life and performance?

Proper storage is critical for maintaining TUBB2A antibody function over time:

• Standard storage recommendations:

  • Store at -20°C for long-term preservation

  • Aliquot upon receipt to avoid repeated freeze-thaw cycles

  • Store in recommended buffer (typically PBS, pH 7.4, containing 0.02% sodium azide as preservative and 50% glycerol)

• Working stock handling:

  • Keep working aliquots at 4°C for up to one month

  • Return to -20°C for longer storage intervals

  • Monitor for signs of degradation (precipitates, loss of activity)

• Shipping and temporary storage considerations:

  • Brief exposure to ambient temperatures during shipping is generally acceptable

  • Use ice packs when transporting between facilities

  • Return to appropriate storage conditions promptly after use

• Stability monitoring:

  • Periodically test activity against a reference sample

  • Document any changes in performance over time

  • Consider including positive controls from initial validation in new experiments

• Contamination prevention:

  • Use sterile technique when handling antibodies

  • Avoid introducing bacteria or fungi that could degrade the antibody

  • Consider adding antimicrobial agents if diluting for longer-term storage