TUBA1A Monoclonal Antibody

What applications are supported by TUBA1A monoclonal antibodies?

The DM1A clone of TUBA1A monoclonal antibody supports a diverse range of applications in molecular and cellular biology research. These include Western blotting (WB), immunohistochemistry (IHC), immunocytochemistry/immunofluorescence (ICC/IF), immunoprecipitation (IP), flow cytometry (FC), and more specialized techniques like Simple Western and CyTOF . The versatility of this antibody makes it particularly valuable for researchers investigating cytoskeletal dynamics across different experimental platforms.

What are the recommended antibody dilutions for different experimental applications?

Optimal dilutions vary significantly depending on the specific application:

• Western Blot: 1:5000

• Immunohistochemistry (paraffin or frozen sections): 1:100-1:500

• Immunocytochemistry/Immunofluorescence: 1:50000-1:100000

• Immunoprecipitation: 1:50-1:100

• Flow Cytometry: 1 μg per million cells

• Simple Western: 1:50

These recommendations serve as starting points, and researchers should perform optimization experiments to determine the ideal concentration for their specific experimental conditions, sample type, and detection method.

Which species show confirmed reactivity with the DM1A clone?

The DM1A clone demonstrates exceptional cross-species reactivity. Confirmed reactive species include Human, Mouse, Rat, Chicken, Bovine, Guinea Pig, Porcine, and Xenopus . This broad species reactivity makes it particularly valuable for comparative studies across different model organisms. Some commercial antibodies may list fewer species (such as only Chicken, Human, Mouse, and Rat) , but broader reactivity has been validated in multiple studies. When working with species not explicitly mentioned, researchers may need to perform preliminary validation experiments.

What is the proper storage protocol to maintain antibody effectiveness?

For optimal long-term stability, store lyophilized antibody at -20°C . After reconstitution, the antibody can be stored at 4°C for one month or aliquoted and stored at -20°C for up to six months . It is critical to avoid repeated freeze-thaw cycles as they can significantly compromise antibody performance. Working with small aliquots is recommended for experiments requiring repeated use over time.

What controls should be included when using TUBA1A antibodies in research?

Rigorous experimental design with appropriate controls is essential when working with TUBA1A antibodies:

• Positive control: Include samples known to express TUBA1A (e.g., brain tissue lysates from mouse or rat)

• Negative control: Omit primary antibody to assess background from secondary antibody

• Loading control: Use when performing quantitative Western blot analysis

• Isotype control: Include mouse IgG1 kappa (matching the DM1A clone's isotype) for immunoprecipitation or flow cytometry

• Secondary antibody optimization: When using HRP-conjugated anti-mouse IgG, dilutions around 1:10000 have demonstrated optimal results

These controls help validate experimental findings and troubleshoot potential issues with antibody specificity or technical execution.

How do I validate TUBA1A antibody specificity for my experimental system?

Validating antibody specificity involves multiple approaches:

• Western blot analysis should show a specific band at approximately 55 kDa (though the calculated molecular weight is 50 kDa)

• Perform BLAST analysis between your species of interest and the immunogen sequence when working with unvalidated species

• Include positive controls from well-characterized cell lines such as SH-SY5Y, Jurkat, or A549 human cell lines

• For novel applications, conduct preliminary titration experiments to optimize signal-to-noise ratio

• Consider RNAi knockdown or knockout controls to confirm antibody specificity in challenging experimental systems

Thorough validation ensures that experimental observations are truly attributable to TUBA1A rather than non-specific binding.

How can TUBA1A antibodies differentiate between wild-type and mutant tubulin in research models?

TUBA1A antibodies can be powerful tools for investigating tubulin mutations, though with important methodological considerations:

Standard commercial TUBA1A antibodies like DM1A typically recognize both wild-type and mutant forms of the protein. To distinguish between them, researchers can employ dual-labeling strategies using FLAG-tagged constructs. By transfecting cells with FLAG-tagged wild-type or mutant TUBA1A and then performing co-immunostaining with anti-FLAG and anti-α-tubulin antibodies, researchers can visualize their differential incorporation into the microtubule network .

In experimental systems, wild-type TUBA1A appears as linear structures that colocalize with the broader α-tubulin cytoskeleton, while mutant forms (particularly C25F, R64W, and R402C) often appear as both linear structures and puncta distributed throughout the cytoplasm. The puncta typically do not colocalize with α-tubulin, indicating failure to incorporate into the microtubule network .

What methodological approaches can detect differences in microtubule stability between wild-type and mutant TUBA1A?

Cold-induced depolymerization assays provide valuable insights into microtubule stability differences:

• Establish fibroblast cultures expressing wild-type or mutant TUBA1A

• Incubate cells on ice for varying time periods (0, 5, 10, 15, and 20 minutes)

• Fix cells and stain with TUBA1A antibody

• Quantify the percentage of cells showing complete microtubule depolymerization

Research has demonstrated that cells expressing certain TUBA1A mutations (R64W, C25F) show significantly higher rates of complete depolymerization after 10 minutes of cold treatment compared to wild-type controls . This approach allows researchers to quantitatively assess how mutations affect fundamental microtubule properties.

How can microtubule density be quantified using TUBA1A antibodies?

Quantitative analysis of microtubule density can reveal important differences between wild-type and mutant TUBA1A:

• Transfect cells with FLAG-tagged wild-type or mutant TUBA1A constructs

• Perform immunofluorescence using both anti-FLAG and anti-α-tubulin antibodies

• Capture high-resolution images under standardized conditions

• Calculate microtubule density by measuring the area occupied by linear FLAG staining divided by the total cellular area

• Compare density measurements between wild-type and mutant conditions

This approach has revealed that mutations affect microtubule incorporation differently, with some mutations (like R64W) permitting higher levels of incorporation than others (like C25F and R402C) .

What factors can affect TUBA1A antibody performance in immunofluorescence applications?

Multiple factors can impact immunofluorescence results when using TUBA1A antibodies:

• Fixation method: Different fixatives (paraformaldehyde, methanol, glutaraldehyde) can affect epitope accessibility

• Dilution optimization: For ICC/IF applications, the DM1A clone may require significant dilution (1:50000-1:100000) for optimal signal-to-noise ratio

• Permeabilization conditions: Tubulin epitopes may be affected by detergent type and concentration

• Blocking solution: BSA or serum concentration affects background signal

• Incubation time and temperature: Primary antibody binding may require overnight incubation at 4°C for optimal results

• Wash stringency: Insufficient washing can lead to high background

Systematic optimization of these parameters is essential for generating clean, reproducible immunofluorescence results.

How should researchers address cross-reactivity concerns with TUBA1A antibodies?

When considering cross-reactivity:

• Recognize that TUBA1A antibodies may cross-react with other alpha-tubulin isotypes due to sequence homology

• When working with non-validated species, perform sequence homology analysis between the immunogen (chicken brain microtubules) and the target species' TUBA1A

• Include appropriate negative controls (tissues not expressing the target or isotype controls)

• Consider Western blot validation before proceeding with more complex applications

• For absolute specificity requirements, consider using complementary detection methods

The DM1A clone has demonstrated reliable specificity across multiple applications and species, but these considerations remain important for rigorous research.

What common technical issues arise in Western blot applications with TUBA1A antibodies?

Researchers may encounter several challenges when using TUBA1A antibodies for Western blotting:

• Molecular weight discrepancy: TUBA1A has a calculated molecular weight of 50 kDa but typically appears at approximately 55 kDa on Western blots

• Transfer conditions: Tubulin proteins may require specific transfer conditions (150 mA for 50-90 minutes to nitrocellulose membranes)

• Blocking optimization: 5% non-fat milk in TBS for 1.5 hours at room temperature has proven effective

• Dilution sensitivity: While 1:5000 is recommended , some experiments may require adjustment

• Signal development: Enhanced chemiluminescence (ECL) detection systems provide optimal visualization

Precise adherence to validated protocols helps ensure reliable and reproducible Western blot results.

How are TUBA1A antibodies being utilized in neurodevelopmental disorder research?

TUBA1A mutations have been implicated in severe neurodevelopmental disorders including lissencephaly and brain malformations . TUBA1A antibodies offer valuable research tools in this context:

• Comparing microtubule network organization in patient-derived fibroblasts versus controls

• Investigating how specific mutations (C25F, R64W, R402C) affect microtubule incorporation and stability

• Visualizing differences in microtubule density between wild-type and mutant conditions

• Quantifying cold-induced depolymerization rates as a measure of microtubule stability

• Examining colocalization patterns of TUBA1A with other cytoskeletal components

These applications provide mechanistic insights into how TUBA1A mutations disrupt normal brain development at the cellular level.

What considerations apply when using TUBA1A antibodies in mass cytometry (CyTOF) applications?

Mass cytometry represents an advanced application for TUBA1A antibodies:

The DM1A clone is CyTOF-ready , enabling researchers to incorporate TUBA1A detection into high-dimensional analyses of cellular phenotypes. When developing CyTOF panels incorporating TUBA1A:

• Metal conjugation should not interfere with the antibody's epitope binding region

• Panel design should account for potential signal spillover

• Intracellular staining protocols require optimization for tubulin detection

• Fixation and permeabilization conditions may differ from traditional flow cytometry

• Appropriate controls (including metal-minus controls) are essential

This emerging application allows researchers to examine TUBA1A expression in the context of dozens of other cellular markers simultaneously.