TUBB1 Recombinant Monoclonal Antibody

What is TUBB1 and what biological functions does it serve?

TUBB1 (Tubulin, Beta 1 Class VI) encodes a specific member of the β-tubulin protein family that contributes to microtubule formation. This protein is a critical component of the cytoskeleton and participates in various cellular processes including intracellular transport, cell division, cell motility, and intracellular organization . While initially characterized in platelets and megakaryocytes, TUBB1 has now been identified in thyroid tissue and other cell types . In thyroid tissue specifically, TUBB1 expression is detected during development (at 8, 10, and 12 GW in humans and E13.5, E15.5, and E17.5 in mice) and continues through adulthood . Dysfunction in microtubule dynamics due to TUBB1 mutations or deficiencies can contribute to diseases such as thyroid dysgenesis associated with congenital hypothyroidism and potentially other disorders .

How are TUBB1 recombinant monoclonal antibodies generated?

TUBB1 recombinant monoclonal antibodies are produced through a multi-step process that begins with acquisition of the TUBB1 antibody genes. These genes are then introduced into suitable host cells where they serve as templates for synthesizing TUBB1 antibodies using a cell-based expression and translation system . Following synthesis, the antibodies undergo purification via affinity chromatography and are thoroughly tested through various assays including ELISA, immunohistochemistry (IHC), and flow cytometry (FC) . This recombinant production method offers significant advantages over traditional antibody production approaches, including enhanced purity, stability, affinity, and specificity, as well as better batch-to-batch consistency .

What are the key differences between TUBB1 recombinant antibodies and traditional monoclonal antibodies?

TUBB1 recombinant monoclonal antibodies offer several advantages over traditional monoclonal antibodies:

These Hi-AffiTM recombinant antibody benefits include increased sensitivity, confirmed specificity, high repeatability, excellent batch-to-batch consistency, sustainable supply, and animal-free production methods .

What are the validated applications for TUBB1 recombinant monoclonal antibodies?

TUBB1 recombinant monoclonal antibodies have been validated for multiple experimental applications with specific recommended dilutions:

The antibodies have been validated on multiple sample types, including human cell lines (K562, Jurkat, A431, HepG2), mouse tissues, and human tissues .

What sample preparation methods are optimal for TUBB1 detection in Western blotting?

For optimal detection of TUBB1 via Western blotting, samples should be prepared using the following methodology:

• Harvest cells or tissues and wash with ice-cold PBS to remove debris and contaminants

• Lyse samples in a buffer containing protease inhibitors to prevent protein degradation (typically RIPA buffer with 1% protease inhibitor cocktail)

• Homogenize tissues thoroughly or lyse cells with gentle agitation

• Centrifuge lysates at 12,000g for 15 minutes at 4°C to remove insoluble material

• Determine protein concentration using Bradford or BCA assay

• Mix 20-50μg of total protein with appropriate amount of laemmli buffer containing a reducing agent

• Heat samples at 95°C for 5 minutes to denature proteins

• Load samples onto 10-12% SDS-PAGE gels alongside appropriate molecular weight markers

• For TUBB1 detection, use antibody dilutions of 1:1000-1:5000 as validated in cell lysates such as K562, where a band of approximately 50 kDa is expected

When interpreting results, note that while the calculated molecular weight of TUBB1 is approximately 50 kDa, some antibodies may detect it at an observed molecular weight of 111 kDa under certain conditions .

How should researchers optimize immunohistochemistry protocols for TUBB1 detection in thyroid tissue?

For optimal immunohistochemistry detection of TUBB1 in thyroid tissue, researchers should follow this methodological approach:

• Fix tissue samples in 4% paraformaldehyde and embed in paraffin

• Section tissues at 4-6μm thickness and mount on positively charged slides

• Deparaffinize sections through xylene and decreasing ethanol series

• Perform antigen retrieval using citrate buffer (pH 6.0) at 95-100°C for 20 minutes

• Block endogenous peroxidase with 3% hydrogen peroxide for 10 minutes

• Apply protein block (5% normal goat serum) for 1 hour at room temperature

• Incubate with primary TUBB1 antibody at dilutions of 1:20-1:200 overnight at 4°C

• Use appropriate detection system (e.g., HRP-conjugated secondary antibody)

• Develop signal with DAB substrate and counterstain with hematoxylin

• As a positive control, include thyroid tissue sections from developmental stages (e.g., 12 GW in humans) where TUBB1 expression has been confirmed in the cytoplasm of thyroglobulin-producing thyrocytes

• For negative controls, omit primary antibody or use thyroid tissue from TUBB1 knockout models

This protocol has been successfully used to demonstrate β1-tubulin expression in the cytoplasm of thyroglobulin (TG)-producing thyrocytes at 12 weeks of gestation in human thyroid tissue .

How can researchers distinguish between TUBB1 and other β-tubulin isoforms in experimental systems?

Distinguishing TUBB1 from other β-tubulin isoforms requires careful experimental design due to the high sequence homology between tubulin family members:

• Antibody selection: Choose recombinant monoclonal antibodies specifically validated against TUBB1 epitopes not shared with other tubulin isoforms. The epitope recognition site is crucial—antibodies targeting the C-terminal region tend to have better specificity, as this region has greater sequence divergence among tubulin isoforms .

• Knockout/knockdown controls: Include TUBB1 knockout or knockdown samples as negative controls. Research has shown that in TUBB1 knockout mice, compensatory changes occur in the expression of other β-tubulin isoforms (Tubb2a, Tubb5, Tubb2b, and Tubb3), which can be used as validation markers .

• Expression pattern analysis: TUBB1 has tissue-specific expression patterns distinct from other tubulin isoforms, being strongly expressed in platelets (CD41+ cells) but also detected in thyroid epithelial cells (EpCAM+). Compare expression in these tissues versus tissues where other β-tubulin isoforms predominate .

• Molecular weight verification: While most β-tubulins have similar molecular weights around 50 kDa, subtle differences in migration patterns on high-resolution gels can help confirm specificity .

• Mass spectrometry: For definitive identification, use immunoprecipitation followed by mass spectrometry to identify the specific peptide sequences unique to TUBB1.

These approaches collectively provide robust verification of TUBB1-specific detection that distinguishes it from other closely related tubulin family members.

What is known about TUBB1 mutations and their impact on protein function and antibody recognition?

TUBB1 mutations have significant impacts on protein function and potentially on antibody recognition:

Three notable TUBB1 mutations associated with thyroid dysgenesis (TD) have been identified: c.35delG, c.163G>A, and c.318C>G . These mutations share several important characteristics:

• Location and functional impact: All three mutations are located in the N-terminal domain needed for guanosine triphosphate (GTP) activity. The c.318C>G and c.35delG mutations create premature stop codons that remove the intermediate and C-terminal domains required for microtubule-associated protein (MAP) binding .

• Evolutionary conservation: The affected amino acids are strictly conserved across species from humans to zebrafish and across all β-tubulins, indicating their functional importance .

• Functional consequences: These mutations lead to non-functional α/β-tubulin dimers that cannot be incorporated into microtubules, disrupting microtubule integrity and impairing thyroid migration and thyroid hormone secretion .

• Antibody recognition implications: Antibodies targeting epitopes in the truncated regions (for c.318C>G and c.35delG mutations) would fail to recognize the mutant proteins. Epitope mapping is therefore crucial when studying TUBB1 variants. Antibodies targeting the N-terminal domain before amino acid 35 would not detect products of the c.35delG mutation .

• Prevalence: In a cohort study of patients with congenital hypothyroidism and thyroid dysgenesis, TUBB1 mutations were found in 1.1% of cases, with 5.2% of patients exhibiting at least one rare functional variant in TUBB1 .

For researchers studying TUBB1 mutations, careful selection of antibodies with epitopes outside the mutated regions is essential for accurate detection of variant proteins.

What compensatory mechanisms occur in TUBB1-deficient systems that researchers should consider?

In TUBB1-deficient systems, several compensatory mechanisms occur that researchers must consider when designing experiments and interpreting results:

• Upregulation of alternative β-tubulin isoforms: Studies in TUBB1 knockout mice (Tubb1−/−) have demonstrated significant increases in expression levels of other β-tubulin isoforms, particularly:

  • Tubb2a, Tubb5, Tubb2b, and Tubb3 show increased expression in E17.5 thyroids of Tubb1−/− mice compared to wild-type

  • Tubb2b and Tubb3 expression levels remain elevated in adult Tubb1−/− mice

• Altered α-tubulin expression: Compensatory changes also affect α-tubulin family members:

  • Tuba3 and Tuba4 expression in Tubb1−/− mouse thyroids were diminished at E17.5 and in adults

  • This pattern mirrors compensatory changes previously described in platelets of Tubb1−/− mice

• Functional consequences: Despite these compensatory changes, they are insufficient to completely rescue the phenotype:

  • Tubb1−/− mice show impaired thyroid migration and thyroid hormone secretion

  • This suggests that other β-tubulin isoforms cannot fully substitute for TUBB1's specialized functions

• Tissue-specific compensation: The pattern of compensation appears to be tissue-specific, with different responses in thyroid tissue compared to platelets and other tissues

These compensatory mechanisms have significant implications for researchers:

• Control experiments should assess the expression of multiple tubulin family members

• Phenotypic analyses should account for partial functional compensation

• Tissue-specific differences in compensation should be considered when extrapolating findings

• Antibody specificity must be carefully validated to avoid cross-reactivity with upregulated isoforms

What are common technical challenges when using TUBB1 recombinant monoclonal antibodies and how can they be addressed?

Researchers frequently encounter several technical challenges when working with TUBB1 recombinant monoclonal antibodies. Here are methodological approaches to address each issue:

When troubleshooting, remember that TUBB1 expression varies by tissue type and developmental stage. For positive controls, consider using platelets or thyroid tissue samples where TUBB1 expression has been well-documented .

How can researchers validate the specificity of TUBB1 recombinant monoclonal antibodies for their particular experimental system?

Validating antibody specificity is critical for reliable research outcomes. Here is a comprehensive methodological approach for validating TUBB1 recombinant monoclonal antibodies:

• Genetic validation approaches:

  • Use TUBB1 knockout/knockdown models: Compare antibody staining in wild-type vs. TUBB1-deficient samples. Complete absence of signal in knockout samples confirms specificity .

  • Overexpression systems: Compare antibody reactivity in cells overexpressing TUBB1 versus control cells to confirm increased signal.

  • siRNA/shRNA knockdown: Demonstrate reduced antibody signal correlating with decreased TUBB1 mRNA levels.

• Biochemical validation approaches:

  • Peptide competition assays: Pre-incubate antibody with immunizing peptide before application to samples; specific binding should be blocked.

  • Western blot analysis: Confirm detection of a single band at the expected molecular weight (approximately 50 kDa) .

  • Immunoprecipitation followed by mass spectrometry: Definitively identify the captured protein as TUBB1.

• Comparative antibody analysis:

  • Test multiple antibodies targeting different TUBB1 epitopes and compare staining patterns.

  • Include antibodies against other β-tubulin isoforms to demonstrate differential staining.

• Tissue/cell-specific validation:

  • Compare staining in tissues with known TUBB1 expression (e.g., platelets, megakaryocytes, thyroid tissue) versus tissues with minimal expression .

  • Correlate protein detection with mRNA expression data from qPCR or RNA-seq.

• Application-specific controls:

  • For IHC/ICC: Include isotype controls and secondary-only controls.

  • For WB: Include recombinant TUBB1 protein as a positive control.

  • For FC: Use isotype controls and FMO (fluorescence minus one) controls.

By implementing this multi-faceted validation approach, researchers can ensure high confidence in the specificity of their TUBB1 antibody for their particular experimental system.

What are the optimal storage and handling conditions to maintain TUBB1 antibody performance over time?

Proper storage and handling of TUBB1 recombinant monoclonal antibodies is essential for maintaining their performance and extending their useful lifespan:

Long-term Storage Recommendations:

• Temperature conditions: Store antibody at -20°C for long-term preservation (up to one year) .

• Aliquoting strategy: Upon receipt, divide the antibody into small single-use aliquots (10-50μL) to minimize freeze-thaw cycles.

• Storage buffer considerations: Most TUBB1 antibodies are supplied in buffer containing stabilizers such as:

  • 50% glycerol to prevent freezing damage

  • 0.02% sodium azide as a preservative

  • PBS at pH 7.2 for optimal protein stability

Working Stock Handling:

• Short-term storage: For frequent use, store working aliquots at 4°C for up to one month .

• Freeze-thaw cycles: Avoid repeated freeze-thaw cycles which can significantly degrade antibody performance .

• Centrifugation: Briefly centrifuge vials after thawing to collect contents at the bottom of the tube.

• Temperature equilibration: Allow refrigerated antibody to reach room temperature before opening to prevent condensation.

Diluted Antibody Handling:

• Diluent composition: Prepare working dilutions in buffer containing:

  • 1% BSA or casein as a carrier protein

  • 0.01% sodium azide as a preservative (if not used for in vivo applications)

  • Appropriate detergent (0.05% Tween-20 for WB applications)

• Storage of diluted antibody: Use freshly diluted antibody when possible; if storage is necessary, keep at 4°C for no more than 7 days.

Quality Control Measures:

• Periodic validation: Test antibody performance regularly using positive control samples.

• Documentation: Maintain records of freeze-thaw cycles, dilution history, and performance observations.

• Lot monitoring: When using recombinant antibodies from different production lots, verify consistent performance.

Following these methodological recommendations will help ensure reliable and reproducible results when working with TUBB1 recombinant monoclonal antibodies over extended periods.

How can TUBB1 recombinant monoclonal antibodies be employed in studying thyroid development and associated disorders?

TUBB1 recombinant monoclonal antibodies offer powerful tools for investigating thyroid development and associated disorders through multiple methodological approaches:

• Developmental expression profiling:

  • Track TUBB1 expression across developmental stages (8-12 GW in humans, E13.5-E17.5 in mice) using immunohistochemistry to correlate with key thyroid developmental events .

  • Co-stain with thyroglobulin and other thyroid markers to identify specific cell populations expressing TUBB1 .

  • Compare expression patterns with other tubulin isoforms to understand unique developmental roles.

• Thyroid dysgenesis investigation:

  • Screen patient samples for TUBB1 mutations associated with thyroid dysgenesis (TD) and congenital hypothyroidism (CH) .

  • Correlate TUBB1 protein expression levels with specific morphological subtypes of TD (ectopia, hemithyroid, hypoplasia) .

  • Analyze how specific mutations (e.g., c.35delG, c.163G>A, c.318C>G) affect antibody binding and protein localization .

• Functional studies in model systems:

  • Use antibodies to track incorporation of wild-type vs. mutant TUBB1 into microtubules in thyroid cell lines.

  • Perform immunofluorescence co-localization with markers of microtubule dynamics during thyroid cell migration.

  • Investigate TUBB1 involvement in thyroid hormone secretion pathways.

• Patient stratification and diagnostics:

  • Develop immunohistochemical panels including TUBB1 antibodies for characterizing thyroid dysgenesis subtypes.

  • Screen developmental defects for TUBB1 expression abnormalities.

  • Correlate TUBB1 expression patterns with clinical outcomes in thyroid disorders.

• Therapeutic development monitoring:

  • Use antibodies to track effectiveness of gene therapy approaches targeting TUBB1 mutations.

  • Monitor changes in TUBB1 expression during treatment of thyroid developmental disorders.

These applications leverage the finding that TUBB1 mutations occurred in 1.1% of patients with congenital hypothyroidism and thyroid dysgenesis in studied cohorts, with particular association with thyroid ectopia rather than athyreosis .

What emerging research areas might benefit from TUBB1 recombinant monoclonal antibodies beyond traditional applications?

Several emerging research areas stand to benefit significantly from TUBB1 recombinant monoclonal antibodies beyond their traditional applications:

• Single-cell protein profiling:

  • TUBB1 antibodies can be incorporated into mass cytometry (CyTOF) or imaging mass cytometry panels to examine heterogeneity in thyroid epithelial populations

  • Spatial transcriptomics combined with TUBB1 immunolabeling could reveal microenvironmental factors influencing β1-tubulin expression

  • These approaches could uncover previously unknown TUBB1-expressing cell populations beyond the documented thyrocytes and platelets

• Developmental biology and organoid research:

  • TUBB1 antibodies can track microtubule dynamics in thyroid organoid systems

  • Live-cell imaging using non-perturbing TUBB1 antibody fragments could monitor microtubule remodeling during organ morphogenesis

  • Compare TUBB1 incorporation patterns between normal and dysgenesis-model organoids

• Extracellular vesicle (EV) characterization:

  • Investigate potential presence of TUBB1 in thyroid-derived extracellular vesicles

  • Examine whether TUBB1 mutations affect EV cargo loading or trafficking

  • Develop EV-based liquid biopsy approaches for thyroid disorders using TUBB1 antibodies

• Intersections with other developmental disorders:

  • Explore TUBB1 expression in neural crest derivatives, which contribute to thyroid development

  • Investigate potential links between TUBB1 mutations and other congenital disorders involving cell migration defects

  • Examine overlaps between TUBB1-associated thyroid dysgenesis and platelet disorders

• Regenerative medicine applications:

  • Monitor TUBB1 expression during directed differentiation of stem cells toward thyroid lineages

  • Track microtubule remodeling in thyroid tissue repair processes

  • Explore TUBB1's role in establishing cell polarity during regenerative processes

• CRISPR-based therapeutic development:

  • Use TUBB1 antibodies to verify successful gene editing outcomes in model systems

  • Monitor specificity of CRISPR approaches targeting TUBB1 mutations

  • Track restoration of normal TUBB1 expression following genetic interventions

These emerging applications highlight the value of highly specific TUBB1 recombinant monoclonal antibodies beyond traditional research applications, potentially opening new avenues for understanding developmental disorders and developing therapeutic interventions.

How can researchers integrate TUBB1 antibody data with other molecular techniques for comprehensive pathway analysis?

Integrating TUBB1 antibody data with other molecular techniques enables comprehensive pathway analysis through a multi-modal approach:

• Multi-omics integration strategies:

  • Combine TUBB1 immunoprecipitation followed by mass spectrometry (IP-MS) with RNA-sequencing to correlate protein interactions and gene expression changes

  • Overlay TUBB1 protein localization data from immunofluorescence with chromatin accessibility maps (ATAC-seq) to identify relationships between microtubule dynamics and transcriptional regulation

  • Integrate TUBB1 expression levels with metabolomics data to reveal connections between microtubule structure and cellular metabolism

• Spatiotemporal analysis approaches:

  • Perform multiplexed immunofluorescence with TUBB1 antibodies alongside markers for cell cycle, differentiation, and other tubulin isoforms

  • Use time-lapse imaging with TUBB1 antibody fragments to track dynamic changes during development or in response to stimuli

  • Correlate TUBB1 expression patterns with cell migration trajectories in developing thyroid tissue

• Genetic perturbation combined with antibody detection:

  • Design CRISPR screens targeting microtubule-associated proteins while monitoring TUBB1 incorporation using antibodies

  • Create mutation series in TUBB1 and use antibodies to track effects on protein localization and function

  • Implement inducible expression systems for TUBB1 variants while monitoring cellular phenotypes with antibody-based detection

• Network analysis methodologies:

  • Use TUBB1 antibodies to identify protein interaction partners through co-immunoprecipitation followed by systematic network analysis

  • Map TUBB1-dependent phosphorylation networks using antibodies against TUBB1 and phospho-specific antibodies for downstream effectors

  • Integrate TUBB1 interactome data with known microtubule regulatory pathways to identify novel connections

• Systems biology implementation:

  • Develop mathematical models incorporating TUBB1 dynamics based on quantitative antibody data

  • Simulate cellular responses to TUBB1 perturbations and validate with experimental antibody-based measurements

  • Use sensitivity analysis to identify critical nodes in TUBB1-dependent pathways

This integrated approach has been effective in elucidating the role of TUBB1 in thyroid development, where researchers combined antibody staining with genetic models, expression analysis, and functional studies to establish TUBB1's critical role in thyroid migration and hormone secretion . Similar methodologies could be applied to understand TUBB1's functions in other tissues and disease contexts.

What key considerations should researchers prioritize when selecting and implementing TUBB1 recombinant monoclonal antibodies in their experimental workflows?

When selecting and implementing TUBB1 recombinant monoclonal antibodies in experimental workflows, researchers should prioritize several key considerations to ensure reliable and meaningful results:

• Experimental application alignment:

  • Match antibody validation profile with intended application (WB, IHC, FC, ICC)

  • Verify the antibody has been tested in your specific application with recommended dilutions (e.g., 1:1000-1:5000 for WB, 1:20-1:200 for IHC)

  • Consider epitope accessibility in your sample preparation method

• Epitope and specificity considerations:

  • Select antibodies targeting epitopes conserved across species if working with multiple models

  • For mutation studies, choose antibodies whose epitopes lie outside the mutated regions

  • Review epitope location regarding functional domains (N-terminal GTP-binding domain vs. C-terminal MAP-binding region)

• Technical validation requirements:

  • Implement appropriate controls (positive, negative, isotype, knockout/knockdown)

  • Verify expected molecular weight (approximately 50 kDa, though may appear at 111 kDa under some conditions)

  • Consider potential cross-reactivity with other β-tubulin isoforms

• Sample-specific optimization:

  • Adjust protocols based on tissue/cell type (thyroid tissue vs. platelets)

  • Consider developmental stage-specific expression patterns

  • Optimize fixation and antigen retrieval methods for tissue-specific requirements

• Data integration planning:

  • Design experiments to facilitate integration with other data types (genomics, transcriptomics)

  • Establish quantification methods appropriate for your research questions

  • Consider how antibody-generated data will fit into larger pathway analyses

By systematically addressing these considerations, researchers can maximize the reliability and scientific value of experiments employing TUBB1 recombinant monoclonal antibodies while avoiding common pitfalls that lead to irreproducible or difficult-to-interpret results.

What are recommended resources for further information on TUBB1 biology and antibody applications?

For researchers seeking to deepen their understanding of TUBB1 biology and antibody applications, the following resources provide valuable specialized information:

• Primary literature on TUBB1 function:

  • Lecine et al. (2000) in Blood (96:1366-73): Foundational paper on TUBB1 function in platelet formation

  • Schwer et al. (2001): Original characterization of Tubb1 knockout mice

  • Recent studies in EMBO Molecular Medicine documenting TUBB1 mutations in thyroid dysgenesis

• Tubulin biology resources:

  • The Human Protein Atlas (proteinatlas.org): Comprehensive tissue expression data for TUBB1

  • UniProt entry (Q9H4B7): Detailed protein information including domains, modifications, and variants

  • STRING database: Protein-protein interaction network for TUBB1

• Antibody validation resources:

  • International Working Group for Antibody Validation (IWGAV) guidelines

  • The Antibody Registry: Database of antibody identifiers and validation information

  • Antibodypedia: User-contributed antibody validation data

• Genetic and clinical resources:

  • OMIM: 612901 - Comprehensive information on TUBB1-related disorders

  • KEGG: hsa:81027 - Pathway information related to TUBB1

  • ClinVar: Database of clinically relevant variants in TUBB1

• Methodological resources:

  • Protocols optimized for tubulin detection in various applications

  • Recommended fixation and antigen retrieval methods for TUBB1 in different tissues

  • Guidelines for distinguishing between tubulin isoforms in experimental systems

• Commercial antibody information:

  • Detailed datasheets from antibody suppliers (Creative Biolabs, Sigma-Aldrich, Cusabio, Boster Bio)

  • Technical support from manufacturers for application-specific optimization

  • Validation data for specific catalog numbers and clones