TUBA3C Antibody

What is TUBA3C and why is it important in cellular research?

TUBA3C (Tubulin Alpha 3C) belongs to the tubulin superfamily and represents one of the major components of microtubules. Along with beta-tubulins, alpha-tubulins form heterodimers that constitute the basic building blocks of microtubules. These intracellular cylindrical filamentous structures are involved in diverse cellular processes including mitosis, intracellular transport, cell movement, and maintenance of cell shape. TUBA3C specifically binds two moles of GTP, one at an exchangeable site on the beta chain and one at a non-exchangeable site on the alpha chain . The significance of studying TUBA3C stems from its fundamental role in cytoskeletal organization and cellular dynamics.

What applications are TUBA3C antibodies suitable for?

TUBA3C antibodies have been validated for multiple research applications:

• Western Blotting (WB): For detecting protein expression levels with observed molecular weight around 50 kDa

• Immunohistochemistry (IHC): For visualizing protein localization in tissue sections

• Immunofluorescence (IF): For subcellular localization studies

• ELISA: For quantitative determination of TUBA3C levels

• Immunoprecipitation (IP): For protein-protein interaction studies

The specific dilution ranges vary by application, with typical recommendations being 1:1000-1:5000 for WB, 1:20-1:200 for IHC, and 1:50-1:200 for IF .

What species reactivity can be expected with TUBA3C antibodies?

Most commercially available TUBA3C antibodies demonstrate cross-reactivity with human, mouse, and rat samples due to the high conservation of tubulin proteins across species . Some antibodies may also exhibit reactivity with samples from additional species such as amphibia, birds, and echinoderms based on sequence homology . When selecting an antibody for your research, verify the validated species reactivity in the product data sheet and consider potential cross-reactivity with other alpha-tubulin isoforms due to sequence similarities.

How can TUBA3C antibodies be used to study post-translational modifications of tubulins?

TUBA3C undergoes various post-translational modifications that affect microtubule dynamics and function. Some antibodies specifically recognize certain modified forms, such as tyrosylated tubulin (Tyr-Tubulin) . For studying post-translational modifications:

• Select antibodies that specifically recognize the modification of interest (detyrosination, acetylation, polyglutamylation)

• Use multiple antibodies targeting different modifications for comparative analysis

• Employ immunofluorescence to visualize the spatial distribution of modified tubulins

• Combine with Western blotting to quantify modification levels

• Consider cell cycle synchronization to capture dynamic modifications at specific phases

Some antibodies, like the YL1/2 clone, specifically recognize the epitope requiring an aromatic residue (tyrosine) at the C terminus with two adjacent negatively charged amino acids (Glu-Glu-Tyr in Tyr-Tubulin) .

What strategies can be employed to differentiate between TUBA3C and other alpha-tubulin isoforms?

Differentiating between highly homologous alpha-tubulin isoforms presents a significant challenge. To achieve isoform specificity:

• Select antibodies raised against unique epitopes, particularly those near the C-terminus where sequence divergence is greater

• Validate antibody specificity using knockout/knockdown cell lines or tissues

• Employ RNA interference to selectively reduce target isoform expression

• Use recombinant expression of tagged TUBA3C as a positive control

• Consider immunoprecipitation followed by mass spectrometry for definitive identification

The antibody specificity should be validated using both positive and negative controls. For TUBA3C, HeLa, HepG2, and Jurkat cell lysates have been used as positive controls for Western blotting .

How can TUBA3C antibodies be applied in studying microtubule dynamics during cell division?

Microtubule dynamics during mitosis can be investigated using TUBA3C antibodies through:

• Time-lapse immunofluorescence microscopy to track microtubule reorganization

• Co-immunostaining with cell cycle markers (e.g., phospho-histone H3)

• Examination of microtubule-associated proteins' co-localization during different mitotic phases

• Combining with drug treatments that affect microtubule polymerization (e.g., nocodazole, taxol)

• Correlative light and electron microscopy for ultrastructural analysis

For these experiments, optimize fixation methods to preserve microtubule structures (often paraformaldehyde is preferred over methanol) and consider live-cell imaging with fluorescently tagged tubulin constructs to complement antibody-based approaches.

What are the optimal storage conditions for maintaining TUBA3C antibody activity?

To maintain antibody functionality:

• Short-term storage (up to 3 months): 4°C is suitable for most antibodies

• Long-term storage: -20°C is recommended for up to one year

• Avoid repeated freeze-thaw cycles by preparing small aliquots before freezing

• Store in recommended buffer conditions (typically PBS with glycerol and preservatives like sodium azide)

Most commercial TUBA3C antibodies are supplied in PBS containing 0.02% sodium azide and 50% glycerol at pH 7.4 . This formulation helps maintain antibody stability during storage by preventing microbial contamination and reducing freezing damage.

What controls should be included when using TUBA3C antibodies in experimental procedures?

For rigorous experimental design, include:

• Positive controls: Cell lines with known TUBA3C expression (HeLa, HepG2, Jurkat)

• Negative controls: Primary antibody omission to assess secondary antibody specificity

• Loading controls: For Western blotting, when not using TUBA3C itself as a loading control

• Isotype controls: Particularly important for flow cytometry applications

• Peptide blocking: To confirm epitope specificity by pre-incubating the antibody with immunizing peptide

• siRNA knockdown samples: To validate antibody specificity

When using TUBA3C as a loading control (common practice), verify that experimental conditions do not alter tubulin expression levels, as some treatments might affect cytoskeletal proteins .

What factors should be considered when optimizing Western blotting protocols for TUBA3C detection?

For optimal Western blot results:

• Sample preparation: Use appropriate lysis buffers that solubilize cytoskeletal proteins effectively

• Protein amount: Load 10-20 μg of total protein for cell lysates

• Gel percentage: 10-12% SDS-PAGE gels are suitable for resolving the ~50 kDa TUBA3C protein

• Transfer conditions: Semi-dry or wet transfer at appropriate voltage to ensure complete protein transfer

• Blocking: 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Antibody dilution: Typically 1:1000-1:5000 for primary antibody incubation

• Incubation time: Overnight at 4°C or 1-2 hours at room temperature

• Detection method: HRP-conjugated secondary antibodies with appropriate chemiluminescent substrate

The observed molecular weight for TUBA3C is approximately 50 kDa, which may differ from the calculated molecular weight of 25,832 Da due to post-translational modifications and protein conformation .

How can background issues in immunohistochemistry with TUBA3C antibodies be mitigated?

To reduce background and improve signal-to-noise ratio:

• Optimize blocking: Use 5-10% normal serum from the same species as the secondary antibody

• Antibody titration: Test multiple dilutions to determine optimal concentration (typically 1:20-1:200 for IHC)

• Antigen retrieval: Compare heat-induced epitope retrieval methods to optimize signal

• Endogenous peroxidase blocking: Use 0.3-3% hydrogen peroxide if using HRP detection systems

• Washing steps: Increase number and duration of washes with appropriate buffers

• Secondary antibody selection: Use highly cross-adsorbed secondary antibodies

• Incubation conditions: Consider lower temperature (4°C) with longer incubation time

For neuronal tissues, which show high TUBA3C expression, specific staining should be localized to the neuronal cytoplasm . Comparison with this expected staining pattern can help distinguish specific signal from background.

What approaches can resolve discrepancies between calculated and observed molecular weights of TUBA3C?

When addressing molecular weight discrepancies:

• Consider post-translational modifications (phosphorylation, acetylation, polyglutamylation) that can increase apparent molecular weight

• Evaluate protein conformation effects on electrophoretic mobility

• Verify running conditions and molecular weight standards

• Use gradient gels to improve resolution around the target molecular weight

• Perform 2D gel electrophoresis to separate different isoforms or modified variants

• Conduct mass spectrometry analysis for definitive molecular weight determination

The calculated molecular weight for TUBA3C is 25,832 Da, while the observed molecular weight in SDS-PAGE is typically around 50-68 kDa . This discrepancy is common for cytoskeletal proteins and doesn't necessarily indicate non-specific binding.

How can cross-reactivity issues with TUBA3C antibodies be evaluated and addressed?

To manage potential cross-reactivity:

• Perform epitope mapping to identify the specific binding region

• Conduct comparative analysis using multiple antibodies targeting different epitopes

• Validate with recombinant expression systems using tagged TUBA3C constructs

• Pre-absorb antibodies with recombinant proteins containing potential cross-reactive sequences

• Use Western blotting with reducing and non-reducing conditions to assess specificity

• Sequence alignment analysis to predict potential cross-reactive proteins

Some TUBA3C antibodies may cross-react with other proteins, including E. coli rec A and oxidized actin under certain circumstances . Understanding these cross-reactivities is essential for accurate data interpretation.

How can TUBA3C antibodies be utilized in studying neurodegenerative diseases?

For neurodegenerative disease research:

• Analyze post-translational modification patterns in disease models compared to controls

• Examine microtubule stability and organization in affected neuronal populations

• Investigate tubulin-binding partners using co-immunoprecipitation with TUBA3C antibodies

• Study the effects of disease-associated mutations on tubulin polymerization

• Evaluate therapeutic compounds targeting microtubule stability using TUBA3C as a marker

Alpha-tubulin is the principal tubulin in morphologically differentiated neurons , making TUBA3C antibodies valuable tools for examining cytoskeletal changes in neurodegenerative conditions where microtubule disruption is implicated.

What methodologies enable the use of TUBA3C antibodies in live-cell imaging applications?

For live-cell imaging:

• Use membrane-permeable probes conjugated to TUBA3C antibody fragments

• Consider microinjection of fluorescently labeled antibodies for short-term imaging

• Employ epitope-tagged TUBA3C constructs for stable cell line generation

• Validate antibody specificity in fixed cells before attempting live-cell applications

• Optimize imaging parameters to reduce phototoxicity while maintaining signal detection

The YL1/2 clone has been used in epitope-tagging procedures to detect proteins tagged with a C-terminal Gly-Gly-Phe epitope , which can be adapted for live-cell imaging applications of TUBA3C dynamics.

How can TUBA3C antibodies be integrated into multi-parameter flow cytometry protocols?

For flow cytometry applications:

• Optimize cell fixation and permeabilization to maintain cellular integrity while enabling antibody access

• Titrate antibody concentrations to determine optimal signal-to-noise ratio

• Use fluorophore-conjugated TUBA3C antibodies compatible with your cytometer configuration

• Include appropriate compensation controls when using multiple fluorophores

• Combine with cell cycle markers to correlate TUBA3C expression with cell cycle phases

• Analyze data using dimensionality reduction techniques for complex multi-parameter datasets