TUBB3 Monoclonal Antibody

What is TUBB3 and why is it used as a research target?

TUBB3 (Tubulin beta-3 chain) is a class III member of the beta tubulin protein family with a molecular weight of approximately 50 kDa. Beta tubulins heterodimerize with alpha tubulins to form microtubules, which are essential cytoskeletal components. TUBB3 is primarily expressed in neurons and plays a crucial role in neurogenesis, axon guidance, and maintenance of neuronal architecture. In adults, TUBB3 expression is largely restricted to neuronal tissues, making it an excellent marker for neuronal cells in various applications. Additionally, aberrant TUBB3 expression has been identified in certain cancer types, particularly intrahepatic cholangiocarcinomas, suggesting its potential utility as a diagnostic biomarker in oncology research . Its tissue-specific expression pattern and involvement in critical cellular processes make it an important target for both neuroscience research and cancer studies.

What applications are TUBB3 monoclonal antibodies validated for?

TUBB3 monoclonal antibodies have been validated for multiple research applications, providing versatility across different experimental platforms. These include Western Blotting (WB) for protein expression analysis, Immunohistochemistry (IHC) for tissue localization studies, Immunocytochemistry/Immunofluorescence (ICC/IF) for cellular localization, Immunoprecipitation (IP) for protein-protein interaction studies, and Flow Cytometry for quantitative cellular analysis . The antibodies have demonstrated particular utility in IHC applications, where they can effectively distinguish TUBB3-expressing tissues in paraffin-embedded sections following appropriate antigen retrieval procedures. Successful application has been documented in various tissue types, including testicular seminoma, thyroid papillary carcinoma, urothelial carcinoma, and non-small cell lung cancer tissues . The diverse range of validated applications makes TUBB3 antibodies valuable tools for both targeted hypothesis testing and exploratory research across multiple experimental contexts.

What is the recommended protocol for TUBB3 detection in immunohistochemistry?

The optimal protocol for TUBB3 detection in immunohistochemistry involves several critical steps that must be followed precisely for successful staining. Based on validated methodologies, the recommended procedure begins with proper tissue fixation and sectioning. For paraffin-embedded sections, heat-mediated antigen retrieval in EDTA buffer (pH 8.0) is crucial for exposing the TUBB3 epitope . Following antigen retrieval, tissue sections should be blocked with 10% goat serum to minimize non-specific binding. The primary antibody incubation should be performed with anti-TUBB3 antibody at a 1:50 dilution overnight at 4°C to ensure adequate binding . For secondary detection, Peroxidase Conjugated anti-species IgG (matching the primary antibody host) should be used with a 30-minute incubation at 37°C . Visualization can be achieved using a DAB (3,3'-diaminobenzidine) chromogen system. This protocol has been successfully applied to various tissue types including testicular seminoma, thyroid papillary carcinoma, and urothelial carcinoma tissues, demonstrating consistent and specific staining of TUBB3-positive structures . Researchers should optimize antibody concentrations based on their specific tissue samples and experimental conditions.

What considerations are important when selecting fluorescent conjugates for TUBB3 immunofluorescence studies?

When designing immunofluorescence experiments targeting TUBB3, the selection of appropriate fluorescent conjugates requires careful consideration of several factors to ensure optimal signal detection and specificity. TUBB3 antibodies are available with various fluorophore conjugations, including CF® dyes that offer exceptional brightness and photostability . The choice of fluorophore should be guided by the experimental setup, including microscope specifications and potential co-staining strategies. Importantly, blue fluorescent dyes such as CF®405S and CF®405M are not recommended for detecting low-abundance targets like TUBB3 in certain tissues, as these dyes typically have lower fluorescence intensity and may yield higher non-specific background compared to longer wavelength fluorophores . For multiplex immunofluorescence studies, selecting fluorophores with minimal spectral overlap is essential. The following table summarizes key properties of different fluorophore options for TUBB3 antibody conjugates:

Researchers should consider the target abundance, tissue autofluorescence characteristics, and potential for multiplexing when selecting the optimal fluorophore conjugate for their specific experimental needs .

How should antibody validation be approached when extending TUBB3 antibody use to untested species or tissues?

Validating TUBB3 antibodies for new species or tissues requires a systematic approach to ensure specificity and reliability of results. While manufacturers typically validate antibodies in specific applications and species (commonly human, mouse, and rat for TUBB3), researchers frequently need to extend their use to additional experimental contexts. When considering cross-reactivity with untested species, sequence homology analysis should be the first step to assess potential binding. For instance, primate tissues are likely to show cross-reactivity with human-validated TUBB3 antibodies due to high sequence conservation . Experimental validation should follow a stepwise approach beginning with positive and negative control tissues. For new tissue types, such as hypothalamus, expression database verification is recommended to confirm expected TUBB3 expression levels before experimental testing . Initial validation should employ multiple detection methods when possible (e.g., Western blot confirmation of IHC findings). Titration experiments to determine optimal antibody concentration in the new context are essential, as sensitivity may differ between tissues. Finally, blocking peptide experiments or knockout/knockdown controls provide the strongest validation of specificity. Documentation of these validation steps is crucial for research reproducibility and may benefit the broader research community facing similar experimental challenges .

How specific is TUBB3 as a marker in cancer diagnosis compared to other markers?

TUBB3 demonstrates distinctive specificity characteristics that position it as a valuable diagnostic marker in certain cancer contexts, particularly for intrahepatic cholangiocarcinomas (CCs). Comparative studies have evaluated TUBB3 against established biliary markers (CK7 and CK19) and malignancy indicators (p53 and MUC1) with revealing results. TUBB3 exhibits moderate sensitivity (50% for peripheral CCs) but exceptionally high specificity for discriminating peripheral CCs from other primary liver tumors . This high specificity stems from the complete absence of TUBB3 expression in hepatocellular carcinomas, biliary premalignant lesions (biliary intraepithelial neoplasias, intraductal papillary neoplasms), peribiliary gland hamartomas, and non-neoplastic biliary epithelium . While CK7 and CK19 show higher sensitivity as biliary markers, they lack the discriminatory power of TUBB3 for peripheral CCs. The following comparative analysis illustrates these differences:

This specificity profile makes TUBB3 particularly valuable in diagnostic panels where distinguishing peripheral CCs from other primary liver tumors is critical . The complementary use of TUBB3 alongside more sensitive but less specific markers can enhance diagnostic accuracy in challenging cases.

What are the known cross-reactivities of TUBB3 monoclonal antibodies across species?

TUBB3 monoclonal antibodies exhibit variable cross-reactivity patterns across species that researchers should consider when designing comparative or translational studies. Commercially available anti-TUBB3 antibodies have been validated for reactivity with human, mouse, and rat tissues as specified in product documentation . The high sequence conservation of tubulin proteins across mammalian species suggests potential broader cross-reactivity, but experimental validation is essential. Customer inquiries and experimental data indicate that antibodies validated for human TUBB3 detection may show cross-reactivity with primate tissues, though this requires confirmation through direct testing . The molecular basis for this cross-reactivity lies in the evolutionary conservation of the TUBB3 protein sequence, particularly in the epitope regions targeted by monoclonal antibodies. When extending use to untested species, researchers should conduct preliminary validation studies including Western blot analysis to confirm specificity at the expected molecular weight (approximately 50 kDa) . Importantly, even with cross-reactivity at the protein level, tissue expression patterns may vary between species, necessitating careful interpretation of comparative results. Researchers planning multi-species studies should consider consulting antibody manufacturers about unpublished cross-reactivity data or participating in innovator programs that support the validation of antibodies in new species applications .

How can researchers distinguish between different beta-tubulin isotypes when using TUBB3 antibodies?

Distinguishing between closely related beta-tubulin isotypes represents a significant challenge in TUBB3 research due to high sequence homology among family members. TUBB3 (class III β-tubulin) shares structural similarities with other beta-tubulin isotypes, necessitating careful antibody selection and experimental design to ensure isotype specificity. The key to selective detection lies in the use of monoclonal antibodies that target unique epitope regions of TUBB3, particularly the C-terminal region where most sequence divergence occurs among tubulin isotypes . These monoclonal antibodies have been specifically developed to recognize TUBB3 without cross-reacting with other beta-tubulin family members. To confirm isotype specificity, researchers should:

• Select antibodies specifically raised against unique TUBB3 peptide sequences rather than full-length protein

• Verify antibody specificity using Western blot analysis of tissues with known differential expression of tubulin isotypes

• Include appropriate positive controls (neuronal tissues with high TUBB3 expression) and negative controls (tissues expressing other beta-tubulin isotypes but minimal TUBB3)

• Consider dual-labeling approaches with antibodies against different tubulin isotypes to directly assess differential expression

The commonly used TUBB3 antibodies (including clones TUBB3/3731 and TUBB3/3732) have been specifically designed to recognize the unique epitopes of class III beta-tubulin, minimizing cross-reactivity with other isotypes . This selectivity enables accurate assessment of TUBB3 expression even in tissues where multiple beta-tubulin isotypes are present.

What are common causes of false-negative results in TUBB3 immunohistochemistry?

False-negative results in TUBB3 immunohistochemistry can stem from multiple methodological factors that researchers should systematically address. One primary cause is inadequate antigen retrieval, which is critical for exposing TUBB3 epitopes masked during fixation. Heat-mediated antigen retrieval specifically using EDTA buffer (pH 8.0) has proven essential for successful TUBB3 detection . Using alternative buffers or insufficient retrieval time/temperature can dramatically reduce antibody binding. Antibody concentration is another critical factor; the recommended dilution for many TUBB3 antibodies in IHC applications is 1:50, and excessive dilution may result in false negatives . Tissue fixation issues, including overfixation or improper fixative choice, can irreversibly mask epitopes. The detection system sensitivity matters significantly; while DAB-based systems work well for tissues with moderate-to-high TUBB3 expression, amplification systems may be needed for tissues with lower expression levels . Storage conditions of both antibodies and tissue sections can compromise detection; freeze-thaw cycles of antibodies or prolonged storage of cut sections before staining may reduce immunoreactivity. Finally, tissue-specific factors must be considered; certain tissues may require protocol modifications, as evidenced by customer inquiries about hypothalamus detection where TUBB3 is highly expressed but may require specific protocol adjustments . Systematic optimization addressing these factors sequentially can resolve most false-negative issues in TUBB3 immunohistochemistry.

How can background staining be minimized when using TUBB3 antibodies in immunofluorescence?

Background staining in TUBB3 immunofluorescence can significantly compromise data quality and interpretation, requiring strategic protocol optimization to achieve clean, specific signals. Several key approaches can minimize nonspecific background when working with TUBB3 antibodies. First, blocking optimization is critical; while standard protocols recommend 10% goat serum , different samples may benefit from alternative blocking agents such as bovine serum albumin (BSA) or specialized commercial blockers that address tissue-specific autofluorescence. Antibody dilution requires careful titration; starting with manufacturer recommendations (typically 1:50 for immunohistochemistry ) and performing dilution series to identify the optimal concentration that maintains specific signal while reducing background. The choice of fluorophore significantly impacts background levels; notably, blue fluorescent conjugates like CF®405S and CF®405M typically yield higher non-specific background than longer wavelength fluorophores and should be avoided for low-abundance TUBB3 detection . Washing procedures should be optimized with multiple, extended PBS-T (PBS with 0.1-0.3% Tween-20) washes between antibody incubations. For tissues with high autofluorescence, treatment with Sudan Black B (0.1-0.3%) or commercial autofluorescence quenchers following secondary antibody incubation can dramatically improve signal-to-noise ratios. Finally, microscope settings should be optimized using appropriate negative controls to identify minimum detector gain settings that eliminate background while preserving specific signal. These combined approaches can substantially enhance the specificity and clarity of TUBB3 immunofluorescence staining.

What protocol modifications are necessary when detecting TUBB3 in different sample types?

Detecting TUBB3 across diverse sample types requires strategic protocol modifications to accommodate the unique characteristics of each specimen. For formalin-fixed, paraffin-embedded (FFPE) tissues, which represent the most common sample type, heat-mediated antigen retrieval in EDTA buffer (pH 8.0) is essential for epitope exposure . The standard blocking with 10% goat serum and primary antibody incubation at 1:50 dilution overnight at 4°C has been validated across multiple cancer tissue types . For frozen tissues, such as mouse hypothalamus samples mentioned in customer inquiries, fixation steps must be modified; brief post-sectioning fixation with 4% paraformaldehyde (10 minutes) followed by reduced antigen retrieval time is typically sufficient . Cell cultures require different approaches; for immunocytochemistry of cultured neurons or cancer cell lines (e.g., A431 cells), fixation with 4% paraformaldehyde for 15 minutes at room temperature followed by permeabilization with 0.1-0.3% Triton X-100 is appropriate . Western blot applications require complete protein denaturation and reduction to expose the TUBB3 epitope effectively. Flow cytometry applications demand special consideration for preserving epitope accessibility during cell preparation; mild fixation and permeabilization protocols are recommended. The detection systems must also be matched to the sample type; chromogenic detection with DAB works well for most FFPE tissues, while fluorescent detection offers advantages for frozen sections and cultured cells where autofluorescence is less problematic . These tailored modifications ensure optimal TUBB3 detection regardless of sample origin.

How should researchers interpret heterogeneous TUBB3 expression in cancer samples?

Heterogeneous TUBB3 expression in cancer samples presents a significant interpretive challenge that requires careful analytical approaches. This heterogeneity, observed in multiple cancer types, is not random but often reflects underlying biological complexity. Studies of intrahepatic cholangiocarcinomas (CCs) reveal that TUBB3 expression is present in 50% of peripheral CCs but only 15% of perihilar CCs, suggesting distinct biological subpopulations even within a single cancer classification . When encountering heterogeneous TUBB3 staining, researchers should implement systematic quantification approaches, including determination of both staining intensity and percentage of positive cells. A modified H-score system (intensity × percentage) can provide semi-quantitative data for comparison across samples. Intratumoral heterogeneity should be documented by analyzing multiple tumor regions, as TUBB3 expression may vary between tumor center and invasive margins. Correlation with clinicopathological features is essential; although current data show no significant differences in clinicopathological features between TUBB3-positive and -negative cases of CCs , this should be analyzed for each cancer type under study. The heterogeneous expression pattern may also serve as a basis for patient stratification in therapeutic response studies, particularly for tubulin-targeting agents. Finally, contextualizing TUBB3 expression by co-staining with other markers (such as CK7, CK19, or p53) can provide deeper insights into the biological significance of heterogeneous expression patterns . This multilayered analytical approach ensures that heterogeneous TUBB3 expression is interpreted within its proper biological and clinical context.

What is the significance of TUBB3 expression in non-neuronal tissues?

The expression of TUBB3 in non-neuronal tissues, particularly in various cancer types, represents an intriguing biological phenomenon with significant research and diagnostic implications. TUBB3, traditionally considered a neuron-specific marker, demonstrates aberrant expression in several non-neuronal malignancies that likely reflects fundamental alterations in cellular differentiation programs. In intrahepatic cholangiocarcinomas, TUBB3 expression in 50% of peripheral cases but only 15% of perihilar cases suggests anatomically distinct pathogenetic mechanisms . This non-neuronal expression appears to be highly selective; TUBB3 is entirely absent in hepatocellular carcinomas, biliary premalignant lesions, and non-neoplastic biliary epithelium, indicating that its expression is not a general feature of malignant transformation but rather a specific characteristic of certain tumor types . The biological significance may involve altered cytoskeletal dynamics, as TUBB3 contributes to microtubule formation and stability differently than other beta-tubulin isotypes. From a diagnostic perspective, this selective expression pattern confers high specificity for discriminating peripheral CCs from other primary liver tumors, making TUBB3 a valuable addition to diagnostic immunohistochemical panels . Additionally, TUBB3 expression in 40% of metastatic colorectal or breast cancers suggests potential utility in identifying the origin of metastatic tumors . Understanding the molecular mechanisms driving ectopic TUBB3 expression in non-neuronal tissues remains an active research area, with potential implications for targeted therapy development and cancer biology more broadly.

How does TUBB3 expression correlate with neuronal differentiation in research models?

TUBB3 expression serves as a critical marker of neuronal differentiation across various research models, providing insights into developmental processes and pathological conditions. In neuronal development studies, TUBB3 expression emerges during early stages of neuronal commitment and increases progressively with maturation, reflecting its important role in neurogenesis and axon guidance . This expression pattern makes TUBB3 antibodies invaluable tools for tracking neuronal differentiation in stem cell models, including embryonic stem cells, induced pluripotent stem cells, and neural progenitor cells undergoing neuronal conversion. The timing of TUBB3 appearance during differentiation protocols serves as a quality control metric, with expression typically beginning after downregulation of pluripotency markers and concurrent with or following early neuronal markers like Nestin. In three-dimensional organoid models, TUBB3 staining reveals the spatial organization of developing neuronal networks, providing structural information beyond simple expression levels. In disease models, particularly neurodevelopmental disorders, alterations in TUBB3 expression patterns can indicate pathological processes; notably, mutations in the TUBB3 gene itself cause congenital fibrosis of the extraocular muscles type 3, a condition where aberrant TUBB3 function disrupts normal axon guidance . Quantitative assessment of TUBB3 expression (by immunofluorescence intensity, Western blot densitometry, or qPCR) enables objective comparison of neuronal differentiation efficiency across experimental conditions, genetic backgrounds, or pharmaceutical interventions. This multifaceted utility establishes TUBB3 as an essential marker in the neuroscience research toolkit.

How can TUBB3 antibodies be utilized in multiplex immunofluorescence studies?

Multiplex immunofluorescence incorporating TUBB3 antibodies offers powerful capabilities for studying complex cellular relationships but requires strategic experimental design to achieve optimal results. This advanced application enables simultaneous visualization of TUBB3 alongside other markers to characterize cell types, signaling pathways, or tissue architecture in a single specimen. When designing multiplex panels including TUBB3, antibody selection is critical; researchers should choose primary antibodies raised in different host species to enable species-specific secondary detection systems . Alternatively, directly conjugated TUBB3 antibodies with appropriate fluorophores eliminate concerns about secondary antibody cross-reactivity. The selection of compatible fluorophores is crucial; TUBB3 antibodies are available with various fluorescent conjugates including CF®488A, CF®568, CF®594, CF®640R, and CF®647, allowing strategic positioning within multiplex panels . Researchers should avoid blue fluorescent conjugates (CF®405S) for TUBB3 detection in low-expression contexts due to higher background interference . For automated multiplex systems using tyramide signal amplification, careful antibody stripping validation between rounds is essential to prevent false co-localization signals. The staining sequence matters significantly; optimal results typically come from detecting lower-abundance targets earlier in the protocol. Analysis of multiplex data requires careful spectral unmixing and compensation when using fluorophores with partially overlapping emission spectra. This approach has proven particularly valuable in cancer heterogeneity studies, neurodevelopmental research, and analysis of tumor microenvironments, where understanding the relationship between TUBB3-expressing cells and their surrounding cellular context provides crucial biological insights.

What are the implications of TUBB3 as a biomarker for therapeutic resistance in cancer?

TUBB3 has emerged as a significant biomarker with implications for therapeutic resistance in multiple cancer types, warranting detailed investigation in translational cancer research. Although not directly addressed in the provided search results, broader research context indicates that aberrant TUBB3 expression correlates with resistance to microtubule-targeting agents, including taxanes and vinca alkaloids, in several malignancies. This resistance mechanism likely stems from TUBB3's unique biochemical properties compared to other beta-tubulin isotypes, resulting in altered microtubule dynamics and drug binding. In intrahepatic cholangiocarcinomas, where TUBB3 expression is observed in 50% of peripheral cases, this biomarker may help stratify patients for treatment selection . The highly specific expression pattern of TUBB3 in certain cancer types but not others (complete absence in hepatocellular carcinomas and biliary premalignant lesions) suggests that its expression represents a specific adaptation rather than a general feature of malignancy . For researchers investigating therapeutic resistance, immunohistochemical analysis of TUBB3 in pre-treatment biopsies could potentially identify patients less likely to respond to standard microtubule-targeting chemotherapies. This application requires standardized TUBB3 detection protocols and scoring systems to enable reliable patient stratification. Furthermore, the biological mechanisms driving TUBB3 upregulation in resistant tumors represent an important research direction, potentially identifying upstream targets for therapeutic intervention to overcome resistance. Combined analysis of TUBB3 with other resistance markers and molecular characteristics may provide more comprehensive predictive models for personalized treatment approaches in oncology.

What cutting-edge techniques are being developed for TUBB3 detection in research applications?

Cutting-edge techniques for TUBB3 detection are expanding research capabilities beyond traditional applications, enabling more sensitive, quantitative, and spatially resolved analyses. While not explicitly detailed in the provided search results, several advanced methodologies warrant consideration based on the evolving landscape of protein detection technologies. Super-resolution microscopy techniques, including Structured Illumination Microscopy (SIM), Stimulated Emission Depletion (STED), and Single-Molecule Localization Microscopy (SMLM), are being applied to TUBB3 detection to resolve subcellular localization at nanoscale resolution, revealing previously inaccessible details of microtubule organization in neuronal processes. Mass cytometry (CyTOF) incorporating metal-tagged TUBB3 antibodies enables high-dimensional single-cell analysis with simultaneous detection of dozens of other markers without fluorescence spectral overlap limitations. Spatial transcriptomics combined with TUBB3 protein detection allows correlation between protein expression and transcriptomic profiles within preserved tissue architecture. Proximity ligation assays (PLA) using TUBB3 antibodies can detect protein-protein interactions involving TUBB3, providing functional insights beyond mere expression. In vivo imaging approaches using near-infrared fluorophore-conjugated TUBB3 antibodies allow longitudinal tracking of TUBB3-expressing structures in animal models. Digital pathology platforms incorporating automated TUBB3 quantification algorithms enable more objective and high-throughput analysis of expression patterns across large tissue cohorts. These advanced techniques are expanding the utility of TUBB3 antibodies beyond conventional applications, offering unprecedented insights into the dynamics, interactions, and functional significance of TUBB3 in both normal physiology and disease states.