TTC26 Antibody

What is TTC26 and why are antibodies against it important for research?

TTC26 is a protein that functions as an intraflagellar transport (IFT) complex B component with specific roles in transporting ciliary cargoes related to ciliary motility and for assembling cilia of full length . The human version has a canonical amino acid length of 554 residues and a protein mass of approximately 64 kDa, with three identified isoforms . TTC26 antibodies are important research tools for studying cilium assembly, ciliary transport mechanisms, and related signaling pathways such as Hedgehog signaling, which are implicated in numerous developmental processes and diseases.

What are the common applications for TTC26 antibodies?

TTC26 antibodies are primarily used in several key applications:

• Western Blotting (WB): For detecting TTC26 protein expression levels and molecular weight confirmation (typically observed at 60-64 kDa)

• Immunofluorescence/Immunocytochemistry (IF/ICC): For visualizing TTC26 subcellular localization, particularly in ciliary structures

• ELISA: For quantitative measurement of TTC26 protein levels

• Immunohistochemistry (IHC): For detecting TTC26 in tissue sections

• Immunoprecipitation (IP): For studying protein-protein interactions involving TTC26

What are the typical specifications of commercial TTC26 antibodies?

Commercial TTC26 antibodies vary in their specifications, but typically include:

How should TTC26 antibodies be validated before experimental use?

Proper validation of TTC26 antibodies should include:

• Western blot analysis using positive control samples (mouse testis tissue, HEK-293 cells, and NIH/3T3 cells are commonly used)

• Testing specificity through genetic approaches (knockout/knockdown controls)

• Immunofluorescence microscopy to confirm expected ciliary localization (hTERT-RPE1 cells are suitable for this purpose)

• Cross-validation with different antibodies targeting distinct epitopes of TTC26

• Confirmation of specificity through recombinant protein expression experiments

• Testing for cross-reactivity against related proteins with similar sequence or structure

What precautions should be taken when handling TTC26 antibodies?

When working with TTC26 antibodies, researchers should consider:

• Storage conditions: Follow manufacturer recommendations, typically -20°C for long-term storage

• Avoiding freeze-thaw cycles: Aliquot antibodies upon receipt

• Buffer compatibility: Some antibodies contain preservatives like ProClin, which is hazardous and should be handled by trained staff only

• Dilution recommendations: Follow supplier-specific guidelines for each application

• Blocking conditions: Optimize to reduce background and non-specific binding

• Incubation times: May vary by application and specific antibody

How can TTC26 antibodies be used to study protein interactions within the IFT complex?

TTC26 antibodies are valuable tools for investigating protein-protein interactions within the intraflagellar transport machinery. Researchers can:

• Use co-immunoprecipitation (co-IP) experiments with TTC26 antibodies to identify interaction partners. For example, studies have demonstrated that TTC26 interacts with IFT46, another component of the IFT-B complex .

• Employ tandem affinity purification (TAP) approaches: Research has utilized TAP-tagged TTC26 in IMCD3 cells to identify interacting proteins. The protocol involves:

  • Extraction using buffer containing 50 mM HEPES (pH 7.5), 150 mM KCl, 1 mM EGTA, 1 mM MgCl₂, 10% Glycerol, 1 mM DTT, and 0.1% NP-40

  • Initial purification with anti-FLAG M2 affinity gel

  • TEV protease cleavage

  • Secondary purification with S-protein agarose

  • Analysis of purified complexes by mass spectrometry

• Compare wild-type versus mutant TTC26 interaction profiles. Research has shown that the C-terminus of TTC26 is critical for its interaction with IFT46, as demonstrated by the hop mouse mutation which impairs this interaction .

What role does TTC26 play in Hedgehog signaling and how can antibodies help elucidate this function?

TTC26 is implicated in Hedgehog (Hh) signaling, and antibodies can help investigate this relationship:

• The hop mouse harbors a nonsense mutation in the Ttc26 gene that impairs Hedgehog signaling . TTC26 antibodies can be used to:

  • Compare TTC26 protein levels and subcellular localization in wild-type versus hop/hop cells

  • Investigate effects on Gli3 processing, as hop/hop MEFs show altered Gli3-F/Gli3-R ratios and defective SAG-induced degradation of Gli3-F

• Experimental approaches include:

  • Western blotting to monitor Gli3 processing (full-length Gli3-F and repressor Gli3-R) in response to Hh pathway activation

  • Combining TTC26 antibodies with antibodies against Hh pathway components (Smo, Gli1/2/3, Ptch1) for co-localization studies

  • Using TTC26 antibodies in rescue experiments (e.g., comparing endogenous mutant protein with overexpressed wild-type TTC26)

What methodological approaches are recommended for studying TTC26 in ciliary function?

For investigating TTC26's role in ciliary function, consider these methodological approaches:

• Live cell imaging of ciliary transport:

  • TTC26-GFP fusion proteins can be used alongside TTC26 antibodies to validate localization

  • Images can be collected at 20 frames per second and analyzed using kymographs generated with ImageJ

• Comparative immunofluorescence:

  • Use acetylated α-tubulin antibodies to mark cilia alongside TTC26 antibodies

  • Fixed cells can be prepared using methanol fixation (-20°C) for optimal ciliary staining

• Ciliary length and morphology analysis:

  • TTC26 is required for assembling cilia of full length

  • Quantitative measurements of ciliary length in control versus TTC26-depleted cells can reveal functional consequences

• Ciliary proteome analysis:

  • Compare ciliary protein composition in wild-type versus TTC26-deficient cells

  • Use TTC26 antibodies for validation of proteomics findings

How can TTC26 antibodies be used in the context of interpretable machine learning approaches?

Recent advancements in computational biology have enabled the use of antibodies in combination with machine learning approaches:

• Matrix completion frameworks can be used to infer unmeasured antibody-antigen interactions :

  • TTC26 antibodies from different studies with partially overlapping characteristics can be integrated

  • The approach allows prediction of how any antibody would interact with any variant

  • This is particularly useful when combining datasets from different laboratories that use different TTC26 antibody clones or epitopes

• Implementation considerations include:

  • Training derived from one dataset may underestimate the true error for predictions in another dataset

  • Correction factors can be applied to address differences between antibody responses in different studies

  • This approach can guide rational design of experimental panels, potentially reducing the number of measurements needed

What are the key considerations when using TTC26 antibodies across different model organisms?

When using TTC26 antibodies across different model systems:

• Epitope conservation must be considered:

  • Mouse and human TTC26 share high sequence homology, making many antibodies cross-reactive

  • For studies in other organisms like C. reinhardtii (where TTC26 is known as DYF13), species-specific antibodies may be required

• Validation strategies across species:

  • For mouse studies: Testis tissue is an excellent positive control

  • For human studies: HEK-293 cells show detectable expression

  • For C. reinhardtii: Specific antibodies against the N-terminal 15 amino acids (MFYSKSRPQHAARTN) have been developed

• Application-specific considerations:

  • Western blot protocols may need optimization for different species (sample preparation, buffer systems)

  • Immunofluorescence may require tissue-specific fixation methods

  • Control samples from the relevant species should always be included

What are common challenges in detecting endogenous TTC26 and how can they be addressed?

Detecting endogenous TTC26 can be challenging due to:

• Expression level variations:

  • TTC26 may be expressed at low levels in some cell types

  • Enrichment of ciliated cells or tissues with abundant cilia (like testis) can improve detection

• Specificity concerns:

  • Use antibodies targeting different epitopes to confirm findings

  • Include proper negative controls (siRNA knockdown, CRISPR knockout when possible)

  • The hop mouse model can serve as a valuable control, as it harbors a nonsense mutation in Ttc26

• Technical optimization strategies:

  • For Western blotting: Optimize protein extraction methods to ensure complete solubilization

  • For immunofluorescence: Consider methanol fixation at -20°C for optimal preservation of ciliary structures

  • For co-IP experiments: Cross-linking may help capture transient interactions

How can researchers differentiate between wild-type and mutant TTC26 using antibodies?

When studying TTC26 mutations (such as in the hop mouse model):

• Epitope selection considerations:

  • The hop mutation produces a truncated protein lacking the C-terminus

  • Antibodies targeting N-terminal epitopes (like AA 5-122) might detect both wild-type and truncated mutant forms

  • C-terminal antibodies (like AA 467-496) would only detect wild-type protein

• Experimental approaches:

  • Western blotting can distinguish wild-type from truncated forms based on molecular weight

  • Immunoprecipitation experiments can assess protein interaction capabilities (e.g., the hop mutation impairs interaction with IFT46)

  • Immunofluorescence can reveal differences in subcellular localization

• Functional validation using mutagenesis:

  • Rescue experiments comparing wild-type and mutant TTC26 (e.g., hop mutation) can confirm antibody specificity

  • Heterologous expression systems can be used to express and detect different TTC26 variants

What emerging research areas might benefit from TTC26 antibody applications?

Several frontier research areas could benefit from TTC26 antibody applications:

• Single-cell analysis of ciliary dynamics:

  • TTC26 antibodies could be used to study ciliary transport at the single-molecule level

  • Super-resolution microscopy techniques would enhance visualization of TTC26 within ciliary subcompartments

• Developmental and disease models:

  • TTC26's role in Hedgehog signaling suggests importance in development and disease contexts

  • Tissue-specific and temporal studies of TTC26 expression and localization could reveal developmental roles

• Therapeutic development:

  • Understanding TTC26 function might reveal new therapeutic targets for ciliopathies

  • Antibody-based screening assays could identify compounds that modulate TTC26 function or ciliary trafficking

• Structural biology:

  • Antibodies could be used to stabilize TTC26 protein complexes for structural studies

  • Fragment antibodies might facilitate crystallization of TTC26 alone or in complex with interacting partners

What methodological improvements could enhance TTC26 antibody research?

Future methodological advancements may include:

• Development of more specific monoclonal antibodies:

  • Currently, most commercially available TTC26 antibodies are polyclonal

  • Isoform-specific monoclonal antibodies would enhance research precision

• Advanced imaging approaches:

  • FRET-based assays using labeled antibodies could reveal dynamic protein interactions

  • Live-cell imaging compatible antibody fragments could track TTC26 movement in real-time

• Integration with other "-omics" approaches:

  • Combining TTC26 antibody-based methods with proteomics, transcriptomics, and genomics data

  • Using machine learning to predict antibody-antigen interactions across diverse experimental settings