TUBB Antibody

What is TUBB and why is it an important target in research?

TUBB (tubulin beta) is a critical component of the cytoskeleton and a member of the tubulin protein family. The canonical human TUBB protein has 444 amino acid residues with a molecular weight of approximately 49.7 kDa and is primarily localized in the cytoplasm. It plays essential roles in cell division and cytoskeletal organization, making it a valuable target for various research applications. TUBB is widely expressed across many tissue types and undergoes post-translational modifications such as phosphorylation .

The protein is known by several other names including beta Ib tubulin, tubulin beta-1 chain, tubulin beta-5 chain, and tubulin beta chain. Due to its high conservation across species, TUBB antibodies often show cross-reactivity with orthologs from multiple organisms including mouse, rat, bovine, frog, and chimpanzee .

What applications are TUBB antibodies commonly used for?

TUBB antibodies demonstrate utility across numerous experimental techniques:

The extensive literature support for these applications is evident from the over 900 publications citing TUBB antibodies in Western blotting and more than 30 publications for immunofluorescence applications .

How should I validate a new TUBB antibody before integrating it into my research?

Proper validation is essential for ensuring reliable results with TUBB antibodies. A comprehensive validation strategy should include:

• Positive control selection: Use tissues or cells known to express TUBB abundantly (e.g., brain tissue, HeLa cells).

• Molecular weight verification: Confirm detection of a band at ~50 kDa in Western blotting, which corresponds to the expected molecular weight of TUBB .

• Comparative analysis: Test multiple antibodies targeting different TUBB epitopes to verify consistency.

• Knockout/knockdown controls: Where possible, include TUBB-depleted samples as negative controls.

• Cross-reactivity assessment: Test the antibody against related tubulin isotypes to evaluate specificity.

• Application-specific validation: For each intended application (WB, IF, IHC), perform separate validation steps as binding properties may differ between applications.

Recent studies have highlighted the importance of comprehensive antibody validation, as unverified antibodies can produce artifactual signals that compromise experimental reproducibility .

What is the recommended Western blotting minimal reporting standard (WBMRS) when publishing TUBB antibody results?

To enhance reproducibility in Western blot analysis using TUBB antibodies, researchers should adhere to the Western blotting minimal reporting standard (WBMRS). This includes documenting:

• Antibody information: Source, catalog number, RRID (Research Resource Identifier), clone for monoclonals, and lot number .

• Blocking conditions: Blocking agent (e.g., BSA, milk), concentration, and duration.

• Primary antibody conditions: Dilution (e.g., 1:2000-1:12000 for TUBB), diluent composition, incubation temperature, and duration .

• Secondary antibody details: Source, specificity, conjugate type, and dilution.

• Washing procedures: Buffer composition, duration, and number of washes.

• Detection method: Enhanced chemiluminescence, fluorescence, or other methods.

• Controls: Description of positive and negative controls used to validate specificity.

• Sample preparation: Lysis buffer composition, protein quantification method, and loading amount.

This additional information adds only about 100 words to manuscripts but significantly improves reproducibility of Western blotting experiments with TUBB antibodies .

How do post-translational modifications affect TUBB antibody binding and epitope accessibility?

Post-translational modifications (PTMs) of TUBB can substantially impact antibody recognition and binding efficacy:

• Phosphorylation: TUBB undergoes phosphorylation, which can alter epitope conformation. Antibodies targeting regions containing phosphorylation sites may show differential binding depending on the phosphorylation status .

• Acetylation: Acetylated forms of tubulin may require specific antibodies for detection. The E7 clone, for example, reacts specifically with beta-tubulin across multiple species and can recognize tubulin in various modification states .

• Ubiquitination: Studies have shown that artifacts in Western blotting are more prominent with antibodies directed against complex epitopes such as post-translational modifications. This is particularly relevant for TUBB, which undergoes ubiquitination .

• Denaturation effects: Sample preparation methods (particularly denaturing conditions) can impact epitope accessibility. Some TUBB antibodies may perform differently in native versus denatured conditions.

When studying PTMs of TUBB, researchers should select antibodies that either specifically recognize the modified form or bind to regions unaffected by the modification of interest.

What are optimal fixation and antigen retrieval methods for immunohistochemistry with TUBB antibodies?

The effectiveness of TUBB antibodies in immunohistochemistry depends heavily on tissue preparation:

Recommended antigen retrieval approaches:

• Heat-induced epitope retrieval (HIER):

  • Primary option: TE buffer (pH 9.0)

  • Alternative: Citrate buffer (pH 6.0)

• Enzyme digestion: Proteinase K can be used but may cause some tissue degradation.

• No retrieval: Some antibodies (like the E7 clone) may work on methanol-fixed specimens without additional retrieval steps .

For optimal results with paraffin-embedded sections, the recommended dilution range for TUBB antibodies is 1:50-1:500, with exact dilution requiring empirical determination for each tissue type .

How can I address non-specific binding and background issues with TUBB antibodies?

Non-specific binding is a common challenge when working with TUBB antibodies. Effective strategies include:

• Optimize blocking:

  • Increase blocking duration (2-3 hours at room temperature or overnight at 4°C)

  • Test different blocking agents (5% BSA may be more effective than milk for phospho-specific antibodies)

  • Include 0.1-0.3% Triton X-100 for membrane permeabilization in IF/ICC applications

• Antibody dilution optimization:

  • For Western blotting, test a broad dilution range (1:2000-1:12000)

  • For IF/ICC, optimal dilutions typically fall between 1:200-1:800

  • Always titrate new antibody lots even if previously optimized

• Control experiments:

  • Include secondary-only controls to identify non-specific secondary binding

  • Use isotype controls (matching the host and isotype of your TUBB antibody)

  • Consider cross-adsorption of secondary antibodies against common species

• Buffer modifications:

  • Add 0.05% Tween-20 to wash buffers

  • Include 5% serum from the secondary antibody host species

  • Consider using commercial background reducers for problematic samples

What controls should I include when using TUBB as a loading control in Western blot experiments?

• Validation of consistent expression:

  • Confirm TUBB expression stability under your experimental conditions

  • Certain treatments or cell states may alter tubulin expression levels

  • Compare TUBB expression with at least one additional control (e.g., GAPDH, actin)

• Loading range verification:

  • Perform a standard curve with various protein amounts to ensure linear detection

  • Typical linear range for TUBB detection extends from 5-50 μg total protein

  • Document signal saturation threshold for your detection system

• Membrane cutting considerations:

  • When cutting membranes to probe for multiple targets, verify complete separation

  • TUBB (50 kDa) may run close to other proteins of interest

  • Record and report the molecular weight markers used for membrane sectioning

• Stripping and reprobing limitations:

  • Assess signal loss after stripping when TUBB is used for membrane reprobing

  • Multiple stripping cycles can reduce TUBB antigenicity

  • Document stripping protocol details in publications for reproducibility

What are the key considerations when using TUBB antibodies across different species?

• Validated cross-reactivity:

  • ABIN2854998 antibody shows confirmed reactivity with hamster, human, mouse, rat, and zebrafish samples

  • The E7 clone has exceptionally broad cross-reactivity from Chlamydomonas to humans

  • Always verify cross-reactivity experimentally rather than relying solely on manufacturer claims

• Epitope conservation analysis:

  • Central regions of TUBB tend to be most conserved across species

  • The ABIN2854998 antibody targets a sequence within the central region of human TUBB

  • Terminal regions may show greater sequence variation between species

• Application-specific performance:

  • Cross-reactivity may vary between applications (e.g., an antibody might work for WB but not IHC in certain species)

  • The E7 clone is particularly useful for cross-species studies as it has been validated for multiple applications across diverse organisms

• Species-adapted protocols:

  • Optimization of antibody concentration may be necessary when switching species

  • Buffer conditions may require adjustment for different species' tissues

What methodological approaches are recommended for studying TUBB post-translational modifications?

Studying TUBB post-translational modifications requires specialized techniques:

• Modification-specific antibodies:

  • Use antibodies that specifically recognize acetylated, phosphorylated, or other modified forms of TUBB

  • The E7 clone can recognize tubulin in various modification states across species

  • Validate specificity using recombinant proteins with defined modification states

• Sample preparation considerations:

  • Phosphatase inhibitors are crucial when studying phosphorylated TUBB

  • Deacetylase inhibitors (e.g., trichostatin A) preserve acetylation states

  • Rapid processing at cold temperatures minimizes modification loss

• Fractionation approaches:

  • Separate soluble and polymerized tubulin fractions to assess modification distribution

  • Use centrifugation-based approaches to isolate microtubule fractions

  • Compare modification patterns between fractions using Western blotting

• Imaging approaches:

  • Super-resolution microscopy can visualize modified TUBB subpopulations within cells

  • Combine with general TUBB antibodies for colocalization studies

  • Use proximity ligation assays to detect specific modified TUBB populations

How can I optimize multiplexed immunofluorescence experiments involving TUBB antibodies?

Multiplexed detection with TUBB antibodies requires careful planning:

• Antibody compatibility:

  • Select TUBB antibodies from different host species than other target antibodies

  • If using multiple rabbit antibodies, consider sequential immunostaining with tyramide signal amplification

  • Verify absence of cross-reactivity between all secondary antibodies

• Signal strength balancing:

  • TUBB typically gives strong signals due to high abundance

  • Dilute TUBB antibodies (1:200-1:800 for IF/ICC) to prevent signal overpowering

  • Adjust exposure times for each channel independently

• Controls for multiplexed experiments:

  • Single-stain controls to establish bleed-through parameters

  • Secondary-only controls for each secondary antibody

  • Isotype controls for each primary antibody

  • Absorption controls with blocking peptides where available

• Sequential staining protocols:

  • For challenging combinations, perform sequential staining with intermediate fixation

  • Document ordering effects if sequential staining is used

  • Consider spectral unmixing for overlapping fluorophores

When using TUBB as a cytoskeletal marker in multiplexed experiments, plan antibody combinations carefully to ensure compatibility and optimal signal-to-noise ratios for all targets.