TTC30A Antibody

What is TTC30A and what is its biological significance in cellular processes?

TTC30A (tetratricopeptide repeat domain 30A) is a critical component of the ciliary segmentation machinery that plays essential roles in cartilage differentiation and renal tubulogenesis . The protein contains tetratricopeptide repeat (TPR) structural motifs with eight of these motifs folding together to produce a single TPR domain, which typically mediates protein-protein interactions and facilitates the assembly of multiprotein complexes . TTC30A functions as a component of the intraflagellar transport (IFT) complex B and is required for the polyglutamylation of axonemal tubulin . It plays a crucial role in anterograde intraflagellar transport, the process by which cilia precursors are transported from the base of the cilium to their incorporation site at the tip . This protein has a calculated molecular weight of 76 kDa (665 amino acids) and an observed molecular weight of 72-76 kDa on SDS-PAGE gels .

What types of TTC30A antibodies are available for research applications?

Based on current commercial offerings, researchers can choose between at least two major types of TTC30A antibodies:

• Rabbit Polyclonal Antibody (e.g., 25352-1-AP from Proteintech)

  • Host/Isotype: Rabbit/IgG

  • Immunogen: TTC30A fusion protein Ag18361

  • Purification method: Antigen affinity purification

• Mouse Monoclonal Antibody (e.g., 68232-1-Ig from Proteintech)

  • Host/Isotype: Mouse/IgG2b

  • Immunogen: TTC30A fusion protein Ag18361

  • Purification method: Protein A purification

Both antibody types show reactivity with human, mouse, and rat samples, making them versatile tools for comparative studies across these species .

What are the validated applications for TTC30A antibodies?

TTC30A antibodies have been validated for multiple experimental applications:

The antibodies have been positively tested in various sample types including A431 cells, HEK-293 cells, HeLa cells, LNCaP cells, mouse testis, rat testis, and hTERT-RPE1 cells for specific applications .

What are the optimal storage conditions for TTC30A antibodies?

For maximum stability and performance, TTC30A antibodies should be:

• Stored at -20°C

• Kept in their provided storage buffer (PBS with 0.02% sodium azide and 50% glycerol, pH 7.3)

• Expected stability: One year after shipment when properly stored

• Aliquoting is generally unnecessary for -20°C storage

• Some formulations (20μL sizes) contain 0.1% BSA as a stabilizer

It's advisable to avoid repeated freeze-thaw cycles to maintain antibody performance and reliability across experiments.

How should I optimize Western blot protocols when using TTC30A antibodies?

For optimal Western blot results with TTC30A antibodies, consider the following methodology:

• Sample preparation:

  • Use validated positive controls such as A431 cells, HEK-293 cells, mouse testis, or rat testis lysates

  • Ensure complete protein denaturation and reduction

• Dilution optimization:

  • For rabbit polyclonal (25352-1-AP): Start with 1:1000 dilution and adjust within the 1:500-1:2000 range

  • For mouse monoclonal (68232-1-Ig): Start with 1:10000 dilution and adjust within the 1:5000-1:50000 range

• Detection considerations:

  • Expect bands at approximately 72-76 kDa

  • Confirm specificity using knockout samples when possible

• Protocol adaptations:

  • Follow manufacturer-specific protocols for optimal results

  • Consider titrating the antibody in each specific testing system to obtain optimal results

The significantly different dilution ranges between the polyclonal and monoclonal antibodies highlight the importance of empirical optimization in your specific experimental system.

What are the key considerations for using TTC30A antibodies in immunofluorescence studies?

When designing immunofluorescence experiments with TTC30A antibodies:

• Cell model selection:

  • hTERT-RPE1 cells are particularly recommended as they efficiently assemble primary cilia suitable for localization studies and phenotype analysis

• Fixation and permeabilization:

  • PFA fixation has been validated in published protocols

  • Consider using Fluoromount-G as mounting medium

• Dilution recommendations:

  • For rabbit polyclonal (25352-1-AP): Use 1:200-1:800 dilution

  • For mouse monoclonal (68232-1-Ig): Use 1:400-1:1600 dilution

• Controls and visualization:

  • Include negative controls (secondary antibody only)

  • Consider co-staining with other cilia markers for proper localization context

  • When possible, include TTC30A knockout samples as specificity controls

How can I optimize immunoprecipitation protocols using TTC30A antibodies?

For successful immunoprecipitation of TTC30A and its interaction partners:

• Starting material:

  • HEK-293 cells have been validated for IP applications

  • Use 1.0-3.0 mg of total protein lysate per experiment

• Antibody amount:

  • Use 0.5-4.0 μg of antibody (recommended for 25352-1-AP)

  • Optimize antibody-to-lysate ratio empirically for your specific cellular context

• Technical considerations:

  • For tagged protein studies, anti-Flag-M2-agarose beads and Flag-peptide have been successfully used in TTC30A research

  • Consider gentle lysis conditions to preserve protein-protein interactions

• Controls:

  • Include IgG isotype control

  • Consider including TTC30A knockout samples when available

What is the relationship between TTC30A and TTC30B paralogues, and how can this be investigated?

TTC30A and TTC30B are paralogues with highly similar nucleotide sequences that are conserved across multiple species. Research has revealed important functional relationships between these paralogues:

How do TTC30A antibodies contribute to our understanding of intraflagellar transport mechanisms?

TTC30A antibodies have facilitated key discoveries about intraflagellar transport (IFT) mechanisms:

• Function in IFT complex B:

  • TTC30A is a core protein of IFT, named for its tetratricopeptide repeat domains

  • TTC30A antibodies have helped confirm its role in anterograde IFT

  • TTC30A is required for polyglutamylation of axonemal tubulin, which affects ciliary function

• Interaction mapping:

  • Immunoprecipitation with TTC30A antibodies has helped identify interaction partners within the IFT-B complex

  • Studies have shown direct interactions with other IFT components

• Research applications:

  • Localization studies using immunofluorescence with TTC30A antibodies help visualize IFT dynamics

  • Western blot analysis can detect changes in TTC30A expression levels under different experimental conditions

  • Co-immunoprecipitation experiments reveal dynamic protein-protein interactions in the IFT machinery

What CRISPR/Cas9 approaches have been used for studying TTC30A, and how can antibodies validate these models?

CRISPR/Cas9 technology has been employed to create sophisticated genetic models for studying TTC30A function:

• Genetic modification strategies:

  • Generation of specific TTC30A and/or TTC30B knockout cell lines in hTERT-RPE1 and HEK293T cells

  • Introduction of FLAG-tags to the N-terminus of either TTC30A or TTC30B genes via homology-directed repair

  • sgRNAs designed using CCTop software with consideration of gene location and off-target effects

• Validation approaches using antibodies:

  • Western blot verification of knockout efficiency using TTC30A antibodies

  • Immunofluorescence to confirm loss of protein localization in knockout models

  • FLAG-tag antibodies for detection of properly modified proteins

• Experimental applications:

  • For interactome identification of TTC30A and TTC30B at endogenous levels

  • Phenotypic analysis of cilia formation and function in knockout models

  • Comparative studies between single and double knockout models to assess functional redundancy

What cell lines are recommended for TTC30A research, and why?

Based on published research, the following cell lines are particularly well-suited for TTC30A studies:

• hTERT-RPE1 cells (CRL-4000, ATCC):

  • Efficiently assemble primary cilia

  • Ideal for localization studies and phenotype analysis

  • Well-established model for ciliary research

  • Successfully used for immunofluorescence with TTC30A antibodies

• HEK293T cells (CRL-3216, ATCC):

  • Recommended for immunoprecipitation and protein complex analysis

  • High transfection efficiency for expression studies

  • Successfully used for CRISPR/Cas9 modification of TTC30A

  • Validated for Western blot applications with TTC30A antibodies

• Additional validated cell types:

  • A431 cells, HeLa cells, and LNCaP cells have shown positive results in Western blot applications

  • Mouse and rat testis tissues provide important in vivo context for TTC30A expression

How can TTC30A antibodies be used to investigate the relationship between cilia formation and disease states?

TTC30A antibodies serve as valuable tools for investigating ciliopathies and related disorders:

• Functional assessment:

  • Immunofluorescence using TTC30A antibodies can reveal defects in ciliary structure and formation

  • Western blot analysis can identify altered expression levels in disease models

  • Co-localization studies can determine proper positioning within ciliary structures

• Research applications:

  • Evaluation of cilia length and morphology in TTC30A-deficient models

  • Assessment of polyglutamylation status of axonemal tubulin using TTC30A antibodies

  • Analysis of IFT complex B integrity in disease states

• Methodological considerations:

  • Combined use of TTC30A and TTC30B antibodies to assess compensation mechanisms

  • Integration with other ciliary markers for comprehensive phenotyping

  • Correlation of molecular findings with functional outcomes in disease models

What controls should be included when using TTC30A antibodies to ensure experimental validity?

Implementing appropriate controls is essential for reliable results with TTC30A antibodies:

• Positive controls for Western blot:

  • A431 cells, HEK-293 cells, mouse testis, and rat testis have been validated

  • Expected molecular weight is 72-76 kDa

• Negative controls:

  • Secondary antibody only controls for immunofluorescence

  • Isotype controls for immunoprecipitation

  • CRISPR/Cas9-generated TTC30A knockout samples when available

• Validation approaches:

  • Comparison of results between polyclonal (25352-1-AP) and monoclonal (68232-1-Ig) antibodies

  • Correlation with mRNA expression data

  • Peptide competition assays to confirm specificity

• Technical considerations:

  • Titration of antibody concentration for each application and cell type

  • Inclusion of loading controls for Western blot

  • Parallel staining with known ciliary markers for co-localization studies

How can I troubleshoot common issues when working with TTC30A antibodies?

When facing challenges with TTC30A antibody experiments, consider these resolution strategies:

• Weak or absent Western blot signal:

  • Increase antibody concentration within recommended ranges

  • Extend primary antibody incubation time (overnight at 4°C)

  • Confirm sample preparation and protein loading

  • Verify expression in your specific cell type with PCR before antibody detection

• Immunofluorescence optimization:

  • Adjust fixation methods (PFA concentration and timing)

  • Optimize permeabilization conditions

  • Increase antibody concentration within recommended ranges

  • Consider signal amplification methods for low-abundance detection

• Immunoprecipitation challenges:

  • Adjust lysis buffer composition to preserve protein-protein interactions

  • Increase antibody amount within the 0.5-4.0 μg range

  • Optimize binding conditions (time, temperature, buffer composition)

  • Consider cross-linking approaches for transient interactions

• Non-specific binding:

  • Increase blocking time and concentration

  • Optimize washing steps (number, duration, buffer composition)

  • Use monoclonal antibody (68232-1-Ig) for potentially higher specificity

  • Include competition controls with immunizing peptide