TBCA Antibody

What is TBCA and what is its biological function?

TBCA (Tubulin-specific chaperone A) is a tubulin-folding protein involved in the early steps of the tubulin folding pathway. It functions as one of four proteins (cofactors A, D, E, and C) involved in directing correctly folded beta-tubulin from folding intermediates. Cofactors A and D are believed to play crucial roles in capturing and stabilizing beta-tubulin in a quasi-native conformation . TBCA is essential for cell viability, and its reduction causes a decrease in soluble tubulin, alterations in microtubules, and G1 cell cycle arrest . Recent research indicates that TBCA primarily receives β-tubulin from the dissociation of pre-existing heterodimers rather than newly synthesized tubulins .

What types of TBCA antibodies are available for research applications?

Researchers have several options when selecting TBCA antibodies:

• Rabbit polyclonal antibodies targeting different regions (e.g., central region, amino acids 30-58)

• Mouse monoclonal antibodies (e.g., clone PAT1A5AT)

• Species reactivity varies, with most antibodies reacting with human, mouse, and rat samples

The selection depends on the specific application and experimental design requirements. Polyclonal antibodies may provide broader epitope recognition, while monoclonal antibodies offer higher specificity for particular epitopes.

What is the molecular weight of TBCA and how does this affect antibody detection?

TBCA has a calculated molecular weight of approximately 12.9-13 kDa (108 amino acids) . This relatively small size can present challenges in some applications, particularly in western blotting where detection of low molecular weight proteins requires appropriate gel concentration and transfer conditions. When working with TBCA antibodies, researchers should expect to observe a band at approximately 13 kDa in western blot applications , though post-translational modifications may sometimes cause slight variations in observed molecular weight.

What are the recommended dilutions for different applications of TBCA antibodies?

Optimal dilutions vary by application and specific antibody. Based on available data, general recommendations include:

These recommendations should serve as starting points for optimization. Sample-dependent variations require titration of the antibody in each testing system to obtain optimal results .

How should TBCA antibodies be stored to maintain optimal activity?

For short-term storage (up to 1 month), TBCA antibodies can be stored at 4°C. For longer periods, store at -20°C . Most formulations contain stabilizers like glycerol (often 50%) and preservatives like sodium azide (0.02-0.09%) . To preserve antibody activity:

• Aliquot antibodies before freezing to prevent repeated freeze-thaw cycles

• For small volume antibodies (e.g., 20 μl sizes), some products contain 0.1% BSA as a stabilizer, and aliquoting may not be necessary

• Always follow manufacturer-specific recommendations, as formulations may vary

What are the recommended protocols for using TBCA antibodies in Western blotting?

When performing Western blot with TBCA antibodies:

• Use an appropriate gel percentage (12-15% is recommended for small proteins like TBCA)

• Transfer to a PVDF or nitrocellulose membrane using standard protocols

• Block with 5% non-fat milk or BSA in TBST

• Apply primary TBCA antibody at recommended dilution (typically 1:300-1:1500)

• Incubate overnight at 4°C or for 1-2 hours at room temperature

• Wash thoroughly with TBST

• Apply appropriate secondary antibody and develop using standard detection methods

Positive controls in Western blot may include human brain tissue and HeLa cells, which have been confirmed to express detectable levels of TBCA .

What factors might affect TBCA antibody specificity and how can they be addressed?

Several factors can influence TBCA antibody specificity:

• Cross-reactivity with related proteins: TBCA is one of several tubulin cofactors. To confirm specificity:

  • Use blocking peptides corresponding to the immunizing antigen

  • Include appropriate negative controls

  • Consider using recombinant TBCA protein fragments as positive controls

• Background signals: To reduce non-specific binding:

  • Optimize blocking conditions (try different blockers and concentrations)

  • Increase washing stringency

  • Titrate antibody concentration to find optimal signal-to-noise ratio

• Sample preparation issues: Ensure complete protein denaturation for Western blotting and appropriate fixation for IHC/IF applications.

How can I validate TBCA antibody specificity in my experimental system?

To validate TBCA antibody specificity:

• Blocking peptide experiments: Pre-incubate the antibody with a 100x molar excess of the protein control fragment for 30 minutes at room temperature before application to samples .

• RNAi validation: Compare staining patterns in cells with normal vs. RNAi-reduced TBCA expression .

• Multiple antibody approach: Use antibodies from different sources or those targeting different epitopes of TBCA.

• Positive and negative controls: Include samples known to express or lack TBCA. Human brain tissue and HeLa cells serve as good positive controls for TBCA expression .

Why might I observe discrepancies in TBCA detection between different experimental approaches?

Discrepancies may arise from:

• Protein conformation differences: In native vs. denatured conditions, epitope accessibility can vary significantly.

• Expression level variations: TBCA expression may differ between cell types and physiological states. For instance, TBCA is abundant in mouse heart insoluble protein extracts .

• Complex formation: TBCA forms complexes with β-tubulin that may mask epitopes or alter antibody accessibility. Studies show that treatments like colchicine can decrease TBCA/β-tubulin complex formation, resulting in increased free TBCA .

• Extraction methods: Different lysis buffers and protocols may yield varying amounts of TBCA, especially given its role in both soluble and cytoskeletal fractions.

How can TBCA antibodies be used to study tubulin heterodimer recycling pathways?

TBCA antibodies are valuable tools for investigating tubulin recycling:

• Co-immunoprecipitation studies: TBCA antibodies can be used to pull down TBCA-tubulin complexes to assess interactions with other cofactors and regulatory molecules .

• Quantification of free vs. complexed TBCA: Western blotting can detect changes in TBCA/β-tubulin complex levels under different conditions. Research shows that colchicine treatment decreases TBCA/β-tubulin complex formation and increases free TBCA, while other anti-mitotic agents like nocodazole or cold shock do not produce this effect .

• Manipulation experiments: Combining TBCA overexpression or knockdown with antibody detection can reveal the protein's role in tubulin heterodimer recycling. Studies indicate that altering TBCA levels, either by RNAi or overexpression, results in decreased tubulin heterodimer levels .

What role does TBCA play in colchicine's mechanism of action, and how can antibodies help elucidate this?

Colchicine is an anti-inflammatory agent that inhibits tubulin polymerization. Research using TBCA antibodies has revealed that:

• Colchicine treatment leads to a decrease in TBCA/β-tubulin complexes and an increase in free TBCA .

• This effect appears specific to colchicine, as it is not observed with other anti-mitotic agents like nocodazole or after cold shock treatment .

• In vitro studies suggest that colchicine inhibits tubulin heterodimer dissociation by TBCE/TBCB, likely by interfering with interactions between TBCE and tubulin dimers, which subsequently leads to free TBCA .

Using antibodies against TBCA, β-tubulin, and other cofactors in immunoprecipitation and immunofluorescence experiments can help map these interaction networks and further elucidate the molecular mechanisms.

How can TBCA antibodies be employed in studies of cell cycle regulation and cytoskeletal dynamics?

TBCA plays a crucial role in maintaining proper tubulin levels, which directly impacts cytoskeletal organization and cell cycle progression:

• Cell cycle studies: TBCA depletion causes G1 cell cycle arrest . Antibodies can track TBCA levels and localization throughout the cell cycle using flow cytometry and immunofluorescence.

• Cytoskeletal reorganization: Using TBCA antibodies in combination with tubulin staining can reveal how TBCA contributes to microtubule dynamics during processes like cell division or migration.

• Disease models: TBCA antibodies can be used to investigate altered expression or function in disease states where microtubule dynamics are disrupted, such as cancer or neurodegenerative disorders.

• Double-labeling experiments: Combining TBCA antibodies with markers for cell cycle phases or cytoskeletal structures can provide insights into temporal and spatial regulation of tubulin folding and recycling.

What is known about TBCA's potential role in disease pathogenesis and how can antibodies contribute to this research?

While TBCA's direct role in disease is still being explored, several research avenues are promising:

• Cancer research: Microtubule dynamics are critical for cell division, and TBCA's role in tubulin homeostasis makes it a potential target for cancer studies. TBCA antibodies can help characterize expression patterns in different tumor types.

• Cardiovascular disease: TBCA is abundant in mouse heart insoluble protein extracts , suggesting a potential role in cardiac function. Antibodies can help investigate TBCA's function in heart tissue and its potential involvement in cardiac pathologies.

• Reproductive biology: TBCA appears in panels of genes associated with female infertility . Immunohistochemistry using TBCA antibodies could help characterize its expression and function in reproductive tissues.

How can TBCA antibodies be integrated into multi-omics approaches for comprehensive cellular studies?

Modern research increasingly combines multiple analytical approaches:

• Immunoprecipitation followed by mass spectrometry: Using TBCA antibodies for immunoprecipitation followed by proteomic analysis can identify novel interaction partners and post-translational modifications.

• ChIP-seq applications: If TBCA is found to interact with nuclear factors, chromatin immunoprecipitation using TBCA antibodies could identify potential regulatory roles.

• Spatial proteomics: Combining TBCA immunofluorescence with multiplexed antibody panels can map its localization relative to other cellular components.

• Systems biology: Integrating TBCA antibody-based data with transcriptomics, proteomics, and functional assays can position TBCA within broader cellular networks and pathways.