TUBB2 Monoclonal Antibody

What is TUBB2B and why is it important for neuroscience research?

TUBB2B is a beta isoform of tubulin that binds GTP and serves as a major component of microtubules. It has significant importance in neurobiology because it is predominantly expressed in developing neurons with highest expression during critical steps of corticogenesis. TUBB2B plays an essential role in neuronal migration and is present in nuclei and nucleoplasm. Research interest in TUBB2B has intensified since mutations in this gene have been identified as a cause of asymmetric polymicrogyria, a complex brain dysgenesis characterized by bilateral, asymmetrical, and anteriorly predominant malformations .

Understanding TUBB2B is crucial for neurodevelopmental research as it functions in microtubule formation, which supports cytoskeletal structure, intracellular transport, and cell division. These processes are fundamental to proper brain development, with disruptions potentially leading to severe neurological disorders .

How do I choose between different types of TUBB2B antibodies for my research?

Selection of the appropriate TUBB2B antibody depends on several experimental factors:

• Target specificity: Determine whether you need an antibody specific to only TUBB2B or one that recognizes multiple beta-tubulin isoforms. Some antibodies like those from Abcam cross-react with both TUBB2A and TUBB2B, which may be advantageous for certain applications .

• Species reactivity: Confirm the antibody's reactivity with your experimental model. For example, the TUBB2B mouse monoclonal antibody from OriGene recognizes human and mouse TUBB2B but has not been tested in other species . Similarly, the RayBiotech rabbit antibody is specifically designed for human samples .

• Application compatibility: Verify the antibody has been validated for your desired application. The mouse monoclonal antibody from OriGene is validated for ELISA, IF, IHC, and WB , while the rabbit polyclonal from RayBiotech is recommended for WB, IHC-P, and IF .

• Clonality considerations: Monoclonal antibodies (like OriGene's AT5B3 clone) offer high specificity to a single epitope, while polyclonal antibodies (like RayBiotech's) may provide stronger signals by binding multiple epitopes.

What are the recommended dilutions for different applications of TUBB2B antibodies?

Optimal dilutions vary by application and specific antibody:

For TUBB2B Mouse Monoclonal Antibody (Clone AT5B3):

• Western blot: 1/250-1/500

• Immunofluorescence: 1/250-1/500

• Immunohistochemistry on paraffin-embedded tissues: 1/50

• ELISA: Application-specific optimization required

For Rabbit Anti-Human TUBB2B (N-term) Antibody:

• Western blotting, IHC-P, and IF applications require optimization, though typically starting dilutions around 1:1000 for WB and 1:200 for IHC and IF are recommended

These dilutions serve as starting points, and optimal concentrations should be determined empirically for each experimental setup to balance signal strength with background minimization.

How should I optimize TUBB2B antibody use in Western blotting protocols?

To optimize Western blotting with TUBB2B antibodies:

• Sample preparation: TUBB2B is abundant in neural tissues, particularly developing brain. For non-neural tissues, enrichment techniques may be required due to lower expression levels. Use fresh samples when possible and include protease inhibitors in lysis buffers to prevent degradation of tubulin proteins.

• Gel selection: Use 10% SDS-PAGE gels for optimal separation, as demonstrated in the experimental protocols with the Abcam antibody .

• Protein loading: For brain tissue, 30-50 μg total protein per lane is typically sufficient, as shown in successful Western blots using mouse brain lysate (50 μg) and NIH-3T3 cell lysate (30 μg) .

• Blocking conditions: 5% non-fat dry milk or BSA in TBST (Tris-buffered saline with 0.1% Tween-20) is generally effective.

• Antibody dilution: Start with the manufacturer's recommended dilution (1:250-1:500 for the OriGene monoclonal antibody or 1:3000-1:5000 for the Abcam antibody ), then adjust based on signal-to-noise ratio.

• Control selection: Include positive controls from tissues known to express TUBB2B (brain tissue or neuronal cell lines). The Abcam antibody was successfully tested with NIH-3T3 and mouse brain lysates .

• Detection method: Choose detection reagents appropriate for your antibody host species (anti-mouse for OriGene's mouse monoclonal or anti-rabbit for RayBiotech's rabbit polyclonal ).

What are the key considerations for using TUBB2B antibodies in immunofluorescence studies?

For optimal immunofluorescence results with TUBB2B antibodies:

• Fixation method: Paraformaldehyde (4%) is generally preferred for preserving microtubule structures. Methanol fixation may be used but can affect epitope availability.

• Permeabilization: Use 0.1-0.3% Triton X-100 to ensure antibody access to intracellular tubulin structures.

• Blocking: 5-10% serum from the species of the secondary antibody source is recommended to reduce non-specific binding.

• Antibody dilution: Start with manufacturer's recommendations (1:250-1:500 for OriGene's antibody ) and optimize as needed.

• Secondary antibody selection: RayBiotech's validation studies used Alexa Fluor 488-conjugated goat anti-rabbit IgG for visualization of their rabbit anti-TUBB2B antibody .

• Nuclear counterstaining: DAPI is commonly used, as demonstrated in RayBiotech's confocal immunofluorescence analysis with HepG2 cells .

• Controls: Include negative controls (primary antibody omission) and positive controls (tissues known to express TUBB2B).

• Image acquisition: Use confocal microscopy when possible for better resolution of subcellular structures, particularly for colocalization studies.

How do I troubleshoot weak or non-specific signals in TUBB2B immunohistochemistry?

When encountering issues with TUBB2B immunohistochemistry:

For weak signals:

• Antibody concentration: Increase antibody concentration incrementally; for the OriGene monoclonal antibody, consider using a more concentrated solution than the recommended 1:50 dilution for paraffin-embedded tissues .

• Antigen retrieval: Optimize antigen retrieval methods (heat-induced epitope retrieval with citrate buffer pH 6.0 or EDTA buffer pH 9.0) to enhance epitope accessibility.

• Incubation conditions: Extend primary antibody incubation time (overnight at 4°C) or adjust incubation temperature.

• Detection system: Switch to a more sensitive detection system like polymer-based methods or tyramide signal amplification.

For non-specific signals:

• Blocking optimization: Increase blocking agent concentration (5-10% normal serum) or duration (1-2 hours).

• Antibody specificity: Confirm antibody specificity using RNAi knockdown controls, as demonstrated in the paper investigating TUBB2B mutations .

• Additional washes: Include more stringent washing steps between antibody incubations.

• Tissue fixation: Overfixation may mask epitopes; adjust fixation time or try different antigen retrieval methods.

• Secondary antibody dilution: Increase dilution of secondary antibody to reduce background.

How can I differentiate between TUBB2A and TUBB2B in my experiments given their high sequence homology?

Differentiating between these highly homologous tubulin isoforms requires careful experimental design:

• Antibody selection: Choose isoform-specific antibodies whenever possible. While some antibodies recognize both isoforms (like Abcam's antibody ), others are specific to TUBB2B (like OriGene's monoclonal antibody ).

• Epitope targeting: Consider antibodies targeting the N-terminal region where sequence differences are more prevalent. The RayBiotech antibody targets the N-terminal region (amino acids 12-39) of human TUBB2B .

• Validation techniques:

  • Use siRNA/shRNA knockdown specific to each isoform to confirm antibody specificity

  • Employ tissues with differential expression patterns (TUBB2B is predominant in developing neurons )

  • Perform immunoprecipitation followed by mass spectrometry for definitive identification

• Cross-validation: When possible, use multiple antibodies targeting different epitopes and compare results.

• Expression analysis: Combine protein detection with mRNA analysis techniques like RT-PCR or RNA-Seq that can employ isoform-specific primers/probes to distinguish between TUBB2A and TUBB2B transcripts.

What methodologies are recommended for studying TUBB2B in neuronal migration disorders?

Based on published research on TUBB2B mutations in neuronal migration disorders:

• Expression analysis: Use in situ hybridization to study spatiotemporal expression patterns during development, as demonstrated in the study of embryonic mice at E14.5 and E16.5 .

• Functional studies: Employ in utero RNA interference to knock down TUBB2B expression. The research showed that using shRNAs targeting either the coding sequence or 3' untranslated region reduced TUBB2B expression by approximately 60%, mimicking the effects of heterozygous mutations .

• Protein functionality assessment: Evaluate the impact of TUBB2B mutations on:

  • GTP binding using structural analysis

  • Protein folding and heterodimer formation with alpha-tubulin

  • Incorporation into microtubules

  • Microtubule dynamics

• Imaging approaches: Utilize advanced neuroimaging techniques (MRI) to characterize brain dysgenesis patterns, which typically show bilateral, asymmetrical, and anteriorly predominant polymicrogyria in patients with TUBB2B mutations .

• Molecular dynamics simulations: Study how mutations affect protein structure and function, particularly for mutations near the GTP/GDP binding site (e.g., S172P, L228P, and F265L) .

• Histopathological analysis: Examine neuronal positioning and layering in affected brain tissues to assess cortical organization defects.

How can I assess the impact of TUBB2B mutations on microtubule dynamics using antibody-based approaches?

To evaluate how TUBB2B mutations affect microtubule dynamics:

• Live-cell imaging: Transfect cells with wild-type and mutant TUBB2B constructs, then use fluorescence recovery after photobleaching (FRAP) or fluorescence correlation spectroscopy to measure microtubule polymerization/depolymerization rates.

• Co-immunoprecipitation: Use TUBB2B antibodies to pull down protein complexes and identify alterations in binding partners caused by mutations.

• Immunocytochemistry: Compare microtubule network morphology between wild-type and mutant TUBB2B-expressing cells. Look for differences in:

  • Microtubule density

  • Organization patterns

  • Post-translational modifications

• GTP hydrolysis assays: Combine with immunoprecipitation using TUBB2B antibodies to isolate tubulin and assess GTP hydrolysis rates of wild-type versus mutant proteins.

• Biochemical fractionation: Separate polymerized (cytoskeleton-associated) from soluble (cytosolic) tubulin pools and use TUBB2B antibodies to quantify distribution between these compartments.

• Proximity ligation assays: Detect protein-protein interactions between TUBB2B and various partners in situ, comparing wild-type and mutant conditions.

The research on TUBB2B mutations demonstrated that mutations like S172P and F265L significantly compromised the ability of TUBB2B to fold properly and form native α/β heterodimers, affecting microtubule assembly and dynamics .

How should I quantify TUBB2B expression levels in different brain regions during development?

For rigorous quantification of TUBB2B expression across brain regions during development:

• Standardized sampling: Collect tissues from precisely defined anatomical regions across consistent developmental timepoints.

• Multiple quantification methods: Employ complementary approaches:

  • Western blotting with TUBB2B antibodies (using the recommended dilutions of 1:250-1:500 for the OriGene antibody )

  • Immunohistochemistry with digital image analysis

  • qRT-PCR for mRNA quantification (as used in the TUBB2B mutation study )

• Reference controls:

  • Use housekeeping proteins (β-actin, GAPDH) for protein normalization

  • Include multiple reference genes for qRT-PCR normalization

  • Process all developmental timepoints in parallel to minimize batch effects

• Statistical analysis: Apply appropriate statistical tests for comparing expression across:

  • Different brain regions

  • Developmental stages

  • Experimental conditions

• Data visualization: Present quantified results as fold changes relative to reference regions/timepoints or as absolute values with appropriate error bars.

• Validation: Cross-validate protein expression results with mRNA data from in situ hybridization or RNA-Seq.

The TUBB2B mutation study employed in situ hybridization to analyze expression patterns in mouse embryos at E14.5 and E16.5, revealing strong expression in the central and peripheral nervous systems, with predominant expression in the cortical plate and a thin layer in the subplate .

What are the key considerations when interpreting TUBB2B immunostaining patterns in pathological samples?

When analyzing TUBB2B immunostaining in pathological contexts:

• Normal expression patterns: Establish baseline TUBB2B distribution in comparable normal tissues. TUBB2B is predominantly expressed in developing neurons with highest expression during corticogenesis .

• Cell-type specificity: Determine which cell populations normally express TUBB2B and whether this pattern is altered in pathological samples.

• Subcellular localization: Assess whether TUBB2B maintains its normal subcellular distribution (usually cytoplasmic and associated with microtubule structures) or shows aberrant localization.

• Control inclusion: Always include appropriate controls:

  • Positive controls (tissues known to express TUBB2B)

  • Negative controls (primary antibody omission)

  • Internal controls (unaffected regions within the same sample)

• Quantitative assessment: Use digital image analysis to quantify:

  • Staining intensity

  • Percentage of positive cells

  • Distribution patterns across tissue layers

• Comparison with established markers: Co-stain with neuronal markers, other cytoskeletal proteins, or cell-cycle markers to provide context for TUBB2B alterations.

• Correlation with clinical data: Integrate immunostaining observations with clinical information, particularly for neurological disorders associated with TUBB2B mutations.

• Technical considerations: Be aware that differences in fixation, processing, and antigen retrieval can affect staining patterns and intensity.

How do I resolve contradictory results between different TUBB2B antibodies in my experiments?

When facing discrepancies between different TUBB2B antibodies:

• Epitope mapping: Identify the specific epitopes recognized by each antibody. The OriGene monoclonal antibody was raised against recombinant human TUBB2B (amino acids 1-445) , while the RayBiotech antibody targets a synthetic peptide from the N-terminal region (amino acids 12-39) .

• Validation status: Review validation data for each antibody, including Western blot bands, immunohistochemistry images, and specificity tests. Both the RayBiotech and Abcam antibodies provide validation images in their product documentation .

• Cross-reactivity assessment: Determine if antibodies cross-react with other tubulin isoforms. The Abcam antibody recognizes both TUBB2A and TUBB2B , which could explain certain discrepancies compared to TUBB2B-specific antibodies.

• Post-translational modifications: Consider whether epitopes might be masked by post-translational modifications in certain contexts.

• Experimental design for resolution:

  • Perform side-by-side comparisons using identical samples and protocols

  • Include genetic approaches (siRNA knockdown, CRISPR knockout) as specificity controls

  • Use recombinant TUBB2B protein as a positive control

  • Test antibodies on samples with known TUBB2B mutations that affect specific domains

• Consult literature: Review publications that have used these antibodies to understand their performance in different experimental settings.

• Technical optimization: Adjust protocols (fixation, antigen retrieval, blocking conditions) for each antibody according to manufacturer recommendations before concluding that results are truly contradictory.

How can TUBB2B antibodies be utilized in studies of GTP-binding and hydrolysis at the tubulin level?

TUBB2B antibodies can be valuable tools for investigating GTP binding and hydrolysis:

• Structural considerations: Research has shown that certain TUBB2B mutations (S172P, L228P, F265L) affect regions involved in GTP binding and hydrolysis. S172 resides in a loop forming part of the guanosine nucleotide-binding site, while L228 and F265 are either in the vicinity of or part of the GTP/GDP binding site .

• Immunoprecipitation-based approaches:

  • Use TUBB2B antibodies to isolate native tubulin complexes

  • Perform GTP binding assays with radioactive or fluorescent GTP analogs

  • Measure GTP hydrolysis rates in immunoprecipitated material

• Comparative analysis: Compare GTP binding/hydrolysis between:

  • Wild-type and mutant TUBB2B proteins

  • Different developmental stages

  • Various cell types or brain regions

• Co-immunoprecipitation: Use TUBB2B antibodies to identify proteins that regulate GTP binding/hydrolysis by co-precipitating with tubulin.

• Conformation-specific antibodies: Consider using antibodies that specifically recognize GTP-bound versus GDP-bound tubulin conformations to complement standard TUBB2B antibodies.

• In vitro reconstitution: Combine purified components with TUBB2B antibodies to monitor GTP hydrolysis during microtubule assembly/disassembly.

Research on TUBB2B mutations demonstrated that alterations near the GTP binding site (like S172P) can disrupt hydrogen bonds and destabilize the GTP pocket, with significant consequences for protein function and microtubule dynamics .

What methodological approaches are recommended for studying TUBB2B-specific chaperone interactions?

Based on research findings about TUBB2B folding and chaperone interactions:

• Co-immunoprecipitation: Use TUBB2B antibodies to pull down protein complexes, followed by analysis of associated chaperones. Research has identified interactions between TUBB2B and tubulin-specific chaperones, particularly Tubulin-specific Chaperone A (TBCA) .

• In vitro translation systems: Study TUBB2B folding using cell-free translation systems supplemented with purified chaperones, as demonstrated in research on TUBB2B mutations that showed disrupted interactions with TBCA .

• Pulse-chase experiments: Monitor the kinetics of TUBB2B folding and incorporation into heterodimers. Research showed that mutations affect this process differentially:

  • p.S172P and p.F265L failed to yield TBCA/β-tubulin intermediates

  • p.L228P and p.F265L showed dramatically reduced native α/β heterodimer formation

  • p.I210T and p.T312M had slightly reduced heterodimer formation

• Structural analysis: Study the binding interfaces between TUBB2B and chaperones using techniques like hydrogen-deuterium exchange mass spectrometry or cryo-electron microscopy.

• Chaperone competition assays: Assess how effectively mutant versus wild-type TUBB2B competes for limiting amounts of chaperones.

• Live-cell imaging: Visualize chaperone-TUBB2B interactions in real-time using fluorescently tagged proteins.

• Expression analysis: Determine whether TUBB2B mutations alter chaperone expression levels as part of compensatory responses.

The research demonstrated that different TUBB2B mutations have distinct effects on chaperone interactions and protein folding, which contributes to their pathogenic mechanisms in neuronal migration disorders .

How should TUBB2B antibodies be used in studying post-translational modifications of tubulins in neurological disorders?

For investigating post-translational modifications (PTMs) of TUBB2B in neurological contexts:

• Modification-specific antibodies: Combine TUBB2B antibodies with antibodies that recognize specific tubulin PTMs (acetylation, tyrosination/detyrosination, polyglutamylation, polyglycylation) in co-labeling experiments.

• Sequential immunoprecipitation: First immunoprecipitate using TUBB2B antibodies, then probe with modification-specific antibodies, or vice versa.

• Mass spectrometry approaches: Immunoprecipitate TUBB2B and analyze using LC-MS/MS to identify and quantify PTMs. Compare modification patterns between:

  • Normal and pathological samples

  • Different developmental stages

  • Various brain regions

• Enzyme inhibition studies: Treat samples with inhibitors of PTM-regulating enzymes (deacetylases, tubulin tyrosine ligase, etc.) before immunostaining with TUBB2B antibodies to assess effects on localization or stability.

• Site-specific mutagenesis: Compare wild-type TUBB2B with mutants that cannot undergo specific modifications to understand their functional importance.

• Drug response studies: Examine how drugs that affect microtubule dynamics or stability influence the PTM pattern of TUBB2B in normal versus pathological conditions.

• Developmental timecourse: Track changes in TUBB2B modifications throughout brain development, particularly during critical periods of neuronal migration and corticogenesis.