TUBB Monoclonal Antibody

What is β-Tubulin (TUBB) and why is it an important research target?

β-Tubulin (TUBB) is one of the primary components of microtubules, which are essential cytoskeletal elements in eukaryotic cells. Microtubules function as constituent parts of the mitotic apparatus, cilia, flagella, and elements of the cytoskeleton . β-Tubulin has a molecular weight of approximately 55,000 Da (55 kDa) and forms heterodimers with α-tubulin to create the fundamental building blocks of microtubules .

β-Tubulin is an important research target for several reasons. First, it serves as a structural marker for studying cytoskeletal dynamics and organization. Second, it's commonly used as a loading control in Western blot experiments, though with limitations in certain tissues . Third, microtubule dynamics are implicated in numerous cellular processes including mitosis, cell motility, and intracellular transport. Finally, alterations in tubulin structure or function are associated with various pathologies, as noted in search results: "Mutation in the gene leads to various neuronal migration disorders such as lissencephaly, pachygyria and polymicrogyria malformations" .

What applications are TUBB monoclonal antibodies suitable for?

TUBB monoclonal antibodies demonstrate versatility across multiple research applications. Based on the available data, these antibodies can be effectively used in:

What isotypes and species reactivity are typical for TUBB monoclonal antibodies?

TUBB monoclonal antibodies are available in different isotypes, each with specific properties that may affect their performance in certain applications:

• IgG1 isotype

• IgG2a Kappa isotype

The isotype of an antibody influences its functional characteristics, including its ability to bind to Fc receptors, activate complement, and its stability under various experimental conditions.

Regarding species reactivity, TUBB monoclonal antibodies often demonstrate broad cross-species recognition. For example, one antibody is reported to react with "Gallus, Monkey, Canine, Hamster, Human, Mouse, Rabbit, Rat, Sheep" . Another antibody is indicated to react with "human, mouse, rat, bovine" . This broad reactivity reflects the high conservation of tubulin structure across species and makes these antibodies versatile tools for comparative studies.

How should TUBB monoclonal antibodies be stored and handled?

Proper storage and handling are crucial for maintaining antibody activity and ensuring consistent experimental results. Based on the search results, recommended conditions include:

• Storage temperature: -20°C or -80°C

• Avoid repeated freeze-thaw cycles

• Shipping condition: Typically shipped on dry ice

TUBB antibodies are typically provided in stabilizing buffer solutions such as:

• Phosphate buffered saline (PBS), pH 7.4, containing 0.02% sodium azide and 50% glycerol

• Buffered aqueous solution without specific additives

The stability information indicates that when properly stored, there is "less than 5% loss rate within the expiration date under appropriate storage condition" . This thermal stability was determined through accelerated thermal degradation testing (incubation at 37°C for 48h) .

For practical laboratory use, consider aliquoting antibodies upon receipt to minimize freeze-thaw cycles and maintain antibody performance throughout the experimental timeline.

How do different clones of TUBB monoclonal antibodies vary in their epitope specificity?

Different clones of TUBB monoclonal antibodies recognize distinct epitopes on the β-tubulin protein, which significantly affects their performance across various applications. From the search results:

• Clone AA2 specifically targets an epitope mapped to amino acids 412-430 of β-tubulin

• Other clones, like C4-2, are mentioned but without specific epitope information

The epitope specificity has profound implications for research applications:

• Structural accessibility: Some epitopes may be masked in certain conformational states or protein complexes, affecting antibody binding in native versus denatured conditions.

• Post-translational modifications: The search results specifically mention an antibody targeting "unmodified" β-tubulin , suggesting that modifications near or at the epitope could interfere with binding.

• Species cross-reactivity: High sequence conservation of epitopes across species results in broad cross-reactivity as reported for these antibodies .

• Functional studies: The search results note that "B2702 peptide binds to β-tubulin and inhibits natural killer (NK) cell cytotoxicity and it influences microtubule polymerization" , highlighting how interactions with specific domains can affect tubulin function.

When selecting a TUBB antibody, researchers should consider whether the epitope is accessible under their experimental conditions and whether it might be affected by the biological processes under study.

What factors influence the reliability of TUBB as a loading control for Western blotting?

While β-tubulin is commonly used as a loading control in Western blot experiments, several factors affect its reliability:

• Tissue-specific expression levels: The search results explicitly caution that "levels of β-Tubulin may not be stable in certain cells. For example, expression of β-Tubulin in adipose tissue is very low and therefore β-Tubulin should not be used as loading control for these tissues" . This variability necessitates validation in each experimental system.

• Molecular weight considerations: β-tubulin has a molecular weight of approximately 50-55 kDa , which may overlap with proteins of interest of similar size, complicating data interpretation.

• Experimental manipulations: Treatments affecting cytoskeletal organization or dynamics may alter β-tubulin levels or extraction efficiency, making it unsuitable as a loading control in such experiments.

• Isoform expression patterns: Humans express multiple β-tubulin isotypes with tissue-specific distribution patterns. The search results list various synonyms: "beta 5-tubulin; beta Ib tubulin; beta-4 tubulin; M40; MGC117247; MGC16435; OK/SW-cl.56; TBB5; TUBB; TUBB1; TUBB5" , indicating this complexity.

• Sample preparation effects: Extraction methods significantly affect the recovery of cytoskeletal proteins, requiring consistent preparation protocols for reliable quantification.

For experiments where β-tubulin may not be suitable, alternative loading controls such as GAPDH, actins, or total protein staining methods should be considered.

How do post-translational modifications of β-tubulin affect antibody recognition?

Post-translational modifications (PTMs) of β-tubulin can significantly impact antibody recognition in multiple ways:

• Direct epitope modification: When modifications occur within or near the epitope sequence, they can directly prevent antibody binding. Search result mentions an antibody targeting "unmodified" β-tubulin, suggesting sensitivity to modifications.

• Conformational alterations: PTMs can induce structural changes that indirectly affect epitope accessibility even when the modification is distant from the epitope.

• Common β-tubulin PTMs with functional significance:

  • Acetylation (particularly on Lys40 of α-tubulin, but also present on β-tubulin)

  • Phosphorylation (affecting microtubule stability)

  • Polyglutamylation and polyglycylation (affecting interactions with microtubule-associated proteins)

  • Tyrosination/detyrosination cycle (regulating microtubule dynamics)

• Functional consequences: The search results note that β-tubulin interactions can "influence microtubule polymerization, which damages cytoskeleton organization and chaperone-like activity of tubulin" , demonstrating how modifications affecting these interactions can impact cellular function.

When studying modified forms of β-tubulin, researchers should either verify that their antibody recognizes the modified form or select modification-specific antibodies where available. Additionally, experimental validation using positive and negative controls with known modification states is essential.

What validation methods are recommended for confirming TUBB antibody specificity?

Comprehensive validation of TUBB antibody specificity is crucial for experimental reliability. Based on established practices in antibody validation and the search results about antibody validation approaches , recommended methods include:

• Western blotting with appropriate controls:

  • Verify single band at expected molecular weight (~50-55 kDa)

  • Include positive controls (cell lines known to express β-tubulin)

  • Include negative controls where feasible (e.g., depleted lysates)

  • Compare results across different antibody clones/vendors

• Immunoprecipitation followed by mass spectrometry:

  • Confirm that immunoprecipitated protein is indeed β-tubulin

  • Can identify potential cross-reactivities with other proteins

  • Provides an orthogonal validation method

• Genetic validation approaches:

  • When possible, test antibody in cells with genetic knockdown/knockout of TUBB

  • Expected result is diminished or absent signal in depleted samples

  • Full sequence assessment, as described in search result , provides additional verification when working with antibody sequences themselves

• Peptide competition assays:

  • Pre-incubate antibody with immunizing peptide (when available)

  • Should result in blocked binding and reduced/eliminated signal

  • Provides direct evidence of binding specificity

• Cross-species validation:

  • Test across multiple species when cross-reactivity is claimed

  • Verify band appears at expected molecular weight in each species

  • Confirms epitope conservation and specificity across species

Search result mentions a "Sequence Validation Percentage" which was developed for validating therapeutic antibody sequences, reflecting the importance of thorough validation approaches in antibody research.

What technical considerations are important when optimizing TUBB immunofluorescence staining?

Optimizing TUBB immunofluorescence staining requires attention to several technical parameters:

• Fixation protocol selection:

  • Different fixation methods affect microtubule preservation and epitope accessibility

  • Methanol fixation (-20°C) preserves microtubule structure while providing permeabilization

  • Paraformaldehyde (4%) followed by detergent permeabilization often provides good results

  • Test multiple fixation conditions to determine optimal protocol for your antibody clone

• Permeabilization considerations:

  • Required for antibody access to intracellular tubulin

  • Typical agents include Triton X-100 (0.1-0.5%) or saponin (0.1-0.2%)

  • Excessive permeabilization can disrupt microtubule integrity

  • Insufficient permeabilization can result in incomplete antibody access

• Antibody concentration optimization:

  • Starting concentration range: 5-20 μg/mL for immunocytochemistry

  • Titrate to determine optimal signal-to-noise ratio

  • Consider longer incubation at lower concentrations (e.g., overnight at 4°C)

• Signal detection and amplification:

  • Direct vs. indirect detection (fluorophore-conjugated primary vs. secondary antibody)

  • Signal amplification through multilayer approaches for low-abundance targets

  • Confocal microscopy for improved resolution of microtubule structures

• Image acquisition parameters:

  • Appropriate exposure settings to avoid saturation/underexposure

  • Z-stack acquisition for complete visualization of 3D microtubule networks

  • Consistent settings across experimental conditions for comparative analysis

• Controls:

  • Secondary-only controls to establish background levels

  • Positive controls (cells with known microtubule patterns)

  • Competitive peptide controls where available

Careful optimization of these parameters will result in specific, high-quality immunofluorescence staining of β-tubulin, revealing detailed microtubule organization within cells.

What is the optimal Western blotting protocol for TUBB monoclonal antibodies?

Based on the search results and standard practices for β-tubulin detection, an optimized Western blot protocol includes:

• Sample preparation:

  • Lyse cells in RIPA buffer containing protease inhibitors

  • Include brief sonication (3-5 pulses) to enhance extraction of cytoskeletal proteins

  • Centrifuge at 12,000-14,000g for 10 minutes at 4°C to remove debris

  • Determine protein concentration (BCA or Bradford assay)

• Gel electrophoresis:

  • Load 10-30 μg total protein per lane

  • Use 10-12% SDS-PAGE (appropriate for 50-55 kDa proteins)

  • Include molecular weight markers

  • Run at 100-120V until adequate separation

• Transfer:

  • Transfer to PVDF or nitrocellulose membrane

  • Use wet transfer for optimal results with cytoskeletal proteins

  • Transfer at 100V for 1 hour or 30V overnight at 4°C

• Blocking:

  • Block with 5% non-fat dry milk in TBST

  • Alternatively, use 3-5% BSA in TBST

  • Block for 1 hour at room temperature

• Primary antibody incubation:

  • Dilute to recommended concentration: 0.004-2 μg/mL or 1-2 μg/mL for Western blotting

  • Incubate overnight at 4°C or 2 hours at room temperature

  • Dilute in blocking buffer

• Washing:

  • Wash 3-5 times with TBST, 5-10 minutes each wash

• Secondary antibody incubation:

  • Use appropriate HRP-conjugated secondary antibody (anti-mouse for these mAbs)

  • Typical dilution 1:2000-1:10,000 in blocking buffer

  • Incubate for 1 hour at room temperature

• Detection:

  • Develop using ECL or similar chemiluminescent detection reagent

  • Expected band at approximately 50-55 kDa

  • Adjust exposure time as needed for optimal signal

This protocol should be optimized for specific experimental conditions, cell types, and detection systems.

How should sample preparation be optimized for different tissue and cell types?

Sample preparation must be tailored to tissue type for optimal TUBB detection:

• Cell culture samples:

  • Direct lysis in RIPA or NP-40 buffer with protease inhibitors

  • Quick processing to prevent tubulin depolymerization

  • Brief sonication can improve cytoskeletal protein extraction

• Brain tissue (high tubulin content):

  • Rapid dissection and freezing to preserve microtubule integrity

  • Homogenization in cold buffer with protease inhibitors

  • Consider including microtubule-stabilizing agents (e.g., taxol, GTP)

  • Higher detergent concentration may be needed (0.5-1% Triton X-100)

• Muscle tissue (fibrous):

  • Mechanical disruption more critical than in soft tissues

  • Higher detergent concentrations required

  • Consider cryogenic grinding for tough samples

  • Filter homogenate to remove connective tissue debris

• Adipose tissue (low tubulin expression):

  • The search results specifically note that "expression of β-Tubulin in adipose tissue is very low"

  • Consider alternative loading controls for these samples

  • Remove lipid layer after centrifugation

  • May require increased sample loading and longer exposure times

• For fixed tissues (IHC applications):

  • Paraffin sections: Heat-induced epitope retrieval (citrate buffer, pH 6.0)

  • Frozen sections: Brief fixation in cold acetone or methanol

  • Recommended antibody concentration: 5-20 μg/mL

  • Include antigen retrieval optimization

• For all sample types:

  • Maintain cold temperatures throughout processing

  • Include protease inhibitor cocktails

  • Process samples rapidly to minimize degradation

  • Consider pilot experiments to determine optimal extraction conditions

Regardless of tissue type, validation with positive controls is essential to confirm successful extraction and detection of β-tubulin.

What approaches are recommended for multiplexing TUBB antibodies with other markers?

Successful multiplexing of TUBB antibodies with other markers requires careful experimental design:

• Antibody selection considerations:

  • TUBB antibodies in this search are mouse monoclonals

  • Pair with antibodies from different host species (e.g., rabbit, goat) for simultaneous detection

  • If multiple mouse antibodies must be used, consider directly conjugated antibodies or sequential staining

• Optimal marker combinations:

  • Cytoskeletal studies: TUBB + actin (phalloidin) + nuclear stain

  • Cell cycle analysis: TUBB + phospho-histone H3 (mitosis marker) + EdU/BrdU (S-phase)

  • Cellular compartments: TUBB + organelle markers (Golgi, ER, mitochondria)

  • Cell type identification: TUBB + cell-type specific markers

• Staining approaches:

  Simultaneous staining:

  • Apply primary antibodies from different species together

  • Wash thoroughly

  • Apply species-specific secondary antibodies

  • Most time-efficient but requires careful cross-reactivity controls

  Sequential staining:

  • Complete first primary-secondary antibody pair

  • Block with excess unconjugated secondary antibody

  • Apply second primary-secondary pair

  • Reduces cross-reactivity but more time-consuming

• Critical controls:

  • Single primary antibody controls (with all secondary antibodies)

  • Secondary-only controls to assess background

  • Absorption controls when available

  • Single-fluorophore samples to set imaging parameters and check for bleed-through

• Fluorophore selection:

  • Choose spectrally separated fluorophores

  • Consider brightness differences (balance exposure settings)

  • Account for autofluorescence of tissue/cells

  • Standard combination: TUBB (green/Alexa 488) + other marker (red/Alexa 594) + nuclear stain (blue/DAPI)

• Imaging considerations:

  • Sequential channel acquisition to minimize bleed-through

  • Consistent exposure settings for quantitative comparisons

  • Z-stack acquisition for 3D relationships

  • High-resolution imaging for co-localization studies

Properly designed multiplexing experiments allow for contextual understanding of β-tubulin distribution and its relationship with other cellular components.

What troubleshooting approaches are effective for common TUBB antibody challenges?

When encountering issues with TUBB antibody performance, these troubleshooting approaches can help resolve common challenges:

• Weak or absent signal in Western blotting:

  • Increase protein loading (10-30 μg → 30-50 μg)

  • Decrease antibody dilution (1:1000 → 1:500)

  • Extend primary antibody incubation (overnight at 4°C)

  • Check extraction buffer (ensure adequate lysis of cytoskeletal components)

  • Verify transfer efficiency (stain membrane for total protein)

  • Consider alternative detection systems with higher sensitivity

• High background in immunofluorescence:

  • Optimize blocking (try different blockers: BSA, normal serum, commercial blockers)

  • Increase washing duration and number of washes

  • Dilute primary antibody further

  • Use centrifugation or filtration to remove antibody aggregates

  • Reduce secondary antibody concentration

  • Include 0.1-0.3% Triton X-100 in antibody diluent

• Non-specific bands in Western blot:

  • Optimize blocking conditions

  • Increase washing stringency (add 0.1% SDS to wash buffer)

  • Try different blocking agents (milk vs. BSA)

  • Pre-absorb antibody with cell/tissue lysate from non-relevant species

  • Verify specificity with knockout/knockdown controls if available

• Poor microtubule morphology in immunostaining:

  • Optimize fixation (test PFA, methanol, and glutaraldehyde)

  • Include microtubule-stabilizing buffers during sample preparation

  • Reduce time between sample collection and fixation

  • Minimize mechanical disruption during processing

  • Consider live-cell fixation to capture native microtubule structures

• Inconsistent loading control results:

  • Remember that "levels of β-Tubulin may not be stable in certain cells"

  • Consider alternative loading controls for problematic tissues

  • Use total protein staining methods (Ponceau S, Coomassie, Stain-free gels)

  • Normalize to multiple housekeeping proteins

• Cross-species reactivity issues:

  • Verify epitope conservation in target species

  • Test antibody on known positive control from target species

  • Consider species-specific antibodies if available

  • Optimize antibody concentration for specific species

Systematic troubleshooting focusing on each step of the protocol will help identify and resolve issues with TUBB antibody applications.

What current trends are emerging in TUBB antibody research applications?

Recent trends in TUBB antibody applications reflect advancing research methodologies and expanding biological questions:

• Integration with high-resolution imaging techniques:

  • Super-resolution microscopy (STORM, PALM, SIM) for nanoscale organization of microtubules

  • Correlative light and electron microscopy (CLEM) for ultrastructural context

  • Live-cell imaging with genetically encoded tubulin markers complementing fixed-cell antibody approaches

• Single-cell applications:

  • Combining TUBB immunostaining with single-cell transcriptomics

  • Monitoring cytoskeletal changes in rare cell populations

  • High-content screening approaches for drug effects on microtubule dynamics

• Therapeutic antibody development:

  • Search result discusses "full validation of therapeutic antibody sequences"

  • Methods like "middle-up LC-QTOF and middle-down LC-MALDI in-source decay (ISD) mass spectrometry" are being applied to antibody validation

  • Development of the "Sequence Validation Percentage" as a quality metric

• Post-translational modification analysis:

  • Increasing focus on specific modified forms of tubulin

  • Development of modification-specific antibodies

  • Combining pan-TUBB antibodies with PTM-specific antibodies to understand modification patterns

• Expansion beyond traditional research applications:

  • Development of monoclonal antibodies as potential therapeutics, as seen with the protective antibody described in search result

  • Although focused on a different target, the principles of characterizing protective effects of monoclonal antibodies may be relevant to TUBB research

These emerging trends highlight the continuing importance of well-validated TUBB antibodies in both basic research and translational applications, with increasing emphasis on specificity, reproducibility, and comprehensive validation.

What best practices should researchers follow for reproducible TUBB antibody experiments?

To ensure reproducible results with TUBB antibodies, researchers should adhere to these best practices:

• Comprehensive antibody documentation:

  • Record complete antibody information: manufacturer, catalog number, lot number, clone name

  • Document the species, isotype, and epitope information when available

  • Maintain detailed records of antibody performance across lots

• Validation for specific applications:

  • Verify antibody performance in each application (WB, IF, IHC, IP)

  • Include appropriate positive and negative controls

  • Perform cross-validation with multiple antibodies when possible

• Detailed methodology reporting:

  • Specify exact antibody concentrations (μg/mL) rather than just dilutions

  • Document all buffer compositions and incubation conditions

  • Describe sample preparation methods in detail, including fixation protocols

  • Report image acquisition parameters for microscopy applications

• Optimization for specific biological systems:

  • Recognize that "optimal working dilutions must be determined by end user"

  • Consider tissue-specific limitations, such as low expression in adipose tissue

  • Develop system-specific protocols rather than applying generic methods

• Appropriate controls:

  • Include loading controls for Western blots (considering TUBB limitations)

  • Use secondary-only controls for immunostaining

  • Include isotype controls to assess non-specific binding

  • Consider competitive peptide controls when available

• Data analysis transparency:

  • Report all image processing steps

  • Use consistent quantification methods

  • Present representative images alongside quantitative data

  • Include statistical analysis appropriate for the experimental design