IFT172 Antibody

What is IFT172 and why is it significant in ciliary research?

IFT172 is the largest protein component of the intraflagellar transport (IFT) complex, playing a crucial role in cilium formation and function. It serves as a key bridging molecule between IFT-A and IFT-B complexes in both anterograde and retrograde IFT trains, making it essential for proper ciliogenesis . The significance of IFT172 in ciliary research stems from its association with several disease variants causing ciliopathies, making it an important target for understanding the molecular basis of these disorders . The protein contains functionally relevant motifs at its C-terminus: a TPR motif involved in IFT-A association and a U-box-like domain involved in ciliary ubiquitination events, both of which are critical for IFT function .

What applications are suitable for IFT172 antibody detection?

Based on validation data, IFT172 antibody (such as the 28441-1-AP antibody) can be effectively used in multiple applications:

For optimal results, each application should be titrated in the specific testing system being used . When conducting immunohistochemistry, antigen retrieval with TE buffer pH 9.0 is suggested, with citrate buffer pH 6.0 as an alternative option .

How should I validate the specificity of an IFT172 antibody?

To validate the specificity of an IFT172 antibody, implement the following methodological approach:

• Western blot analysis: Confirm a single band at the expected molecular weight (approximately 180 kDa observed, compared to the calculated 198 kDa) . Use positive control samples such as mouse testis tissue, rat testis tissue, or mouse brain tissue where IFT172 is known to be expressed .

• Negative controls: Include samples where IFT172 is knocked down or knocked out using siRNA or CRISPR-Cas9 technology. The signal should be significantly reduced or absent in these samples.

• Peptide competition assay: Pre-incubate the antibody with the immunogen peptide (IFT172 fusion protein in the case of 28441-1-AP) before application to the sample. This should abolish specific binding.

• Subcellular localization assessment: In immunofluorescence studies, verify that the staining pattern is consistent with the known localization of IFT172 in ciliary structures.

• Cross-validation with multiple antibodies: If possible, use additional antibodies targeting different epitopes of IFT172 to confirm consistent results.

• Assessment in multiple model systems: Verify reactivity across different species (human, mouse, and rat samples) to confirm cross-reactivity claims .

What are the optimal storage conditions for maintaining IFT172 antibody activity?

For maximum preservation of IFT172 antibody activity, follow these evidence-based storage recommendations:

• Temperature: Store at -20°C for long-term preservation. The antibody remains stable for one year after shipment when stored properly .

• Buffer composition: The optimal storage buffer contains PBS with 0.02% sodium azide and 50% glycerol at pH 7.3, which provides stability and prevents microbial contamination .

• Aliquoting considerations: For antibodies in the storage buffer described above, aliquoting is generally unnecessary for -20°C storage, which simplifies laboratory workflow .

• Working solution handling: After thawing for use, keep the antibody on ice and return to -20°C storage promptly after use to minimize freeze-thaw cycles.

• Contamination prevention: Use sterile technique when handling the antibody to prevent microbial contamination.

• BSA content: Note that smaller volume formats (20μl) may contain 0.1% BSA for additional stability .

How can IFT172 antibody be used to investigate ciliopathies?

IFT172 antibodies serve as valuable tools for investigating ciliopathies through these methodological approaches:

• Mutation-specific analysis: Several missense variants from ciliopathy patients map to the interface between TPRs and the C-terminal domain of IFT172 . Use IFT172 antibodies to assess protein expression levels, localization, and interactions in patient-derived cells compared to controls.

• Functional assessment of pathogenic variants: The IFT172 ciliopathy variant C1727R maps to helix α2 in the U-box domain . Using antibodies directed against IFT172 and interacting partners, researchers can evaluate how this and other mutations affect protein function, stability, and complex formation.

• Analysis of retrograde IFT defects: The FLA11 mutation in the TPR motif of IFT172 has been linked to impaired association with IFT-A, leading to retrograde IFT defects . IFT172 antibodies can help visualize this phenotype through immunofluorescence studies showing accumulation of IFT proteins at the ciliary tip.

• Quantitative assessment of expression levels: Homozygous deletion of the U-box domain in IFT172 leads to reduced protein expression levels and ciliogenesis defects . IFT172 antibodies enable quantitative Western blot analysis to measure these changes.

• Comparative studies across ciliopathy models: Apply IFT172 antibodies in immunostaining protocols across different ciliopathy models to identify common mechanisms of pathogenesis.

What approaches can reveal IFT172 interactions with other IFT components?

To investigate IFT172 interactions with other IFT components, consider these methodological strategies:

• Co-immunoprecipitation (Co-IP): Use IFT172 antibodies to pull down the protein complex and analyze co-precipitated partners, particularly focusing on IFT-A components like IFT144 and IFT140 that are known to interact with the C-terminal region of IFT172 .

• Proximity ligation assay (PLA): This technique can visualize protein-protein interactions in situ with high sensitivity. Use IFT172 antibody in combination with antibodies against potential interacting partners such as IFT-B subunits IFT57/IFT80 (N-terminal interactions) or IFT-A subunits IFT144/IFT140 (C-terminal interactions) .

• Immunofluorescence co-localization: High-resolution microscopy techniques such as STORM or expansion microscopy can be used to visualize the co-localization of IFT172 with other IFT components at the proximal portion of the flagellum .

• Structure-guided interaction analysis: Based on the crystal structure information of IFT172's C-terminal region , design experiments to test specific interaction surfaces. Use mutated versions of IFT172 (e.g., FLA11 mutation or D1605E human ciliopathy variant) and assess their impact on interactions.

• In vitro binding assays: Use purified recombinant proteins together with antibody detection to quantify direct interactions between IFT172 and other IFT components.

How can antibodies be employed to investigate the ubiquitination activity of IFT172?

To study the U-box domain-dependent ubiquitination activity of IFT172, implement these antibody-based approaches:

• Detection of auto-ubiquitination: Use IFT172 antibodies in combination with ubiquitin antibodies to detect auto-ubiquitination events in Western blot analysis. IFT172 has demonstrated auto-ubiquitination activity in vitro when combined with E1 and E2 enzymes .

• Mutation analysis: Analyze the impact of mutations in key residues of the U-box domain (I1688, F1715, P1725) on ubiquitination activity. The P1725A mutation has been shown to abolish ubiquitination activity . Use antibodies to detect both IFT172 and ubiquitin modifications in these mutants.

• E2 enzyme specificity assay: IFT172 shows ubiquitination activity specifically with members of the UbcH5 E2 enzyme family . Use antibodies to detect interaction partners and ubiquitination patterns in assays with different E2 enzymes.

• Structural inhibition assessment: The U-box domain interface involved in E2 binding appears to be occluded in the HsIFT172C2 crystal structure . Design experiments using antibodies that recognize different conformational states to investigate this structural inhibition.

• In vivo ubiquitination analysis: Employ IFT172 antibodies in immunoprecipitation experiments followed by ubiquitin detection to analyze the mono- and poly-ubiquitination patterns in cellular contexts.

What advanced imaging approaches can enhance IFT172 localization studies?

For high-resolution visualization of IFT172 localization, implement these advanced imaging methodologies:

• Super-resolution microscopy: Techniques like STORM (Stochastic Optical Reconstruction Microscopy) can provide nanoscale resolution of IFT172 distribution, revealing its selective association with specific doublet microtubules (DMTs) in the axoneme .

• Ultrastructure expansion microscopy (U-ExM): This technique offers improved resolution compared to conventional immunofluorescence microscopy while allowing molecular identification through antibody staining. It has been successfully used to characterize IFT distribution at the proximal portion of the flagellum .

• Correlative light and electron microscopy (CLEM): Combine IFT172 immunofluorescence with electron microscopy to correlate protein localization with ultrastructural features.

• Volume electron microscopy: This approach has revealed IFT-like densities along microtubule doublets in the proximal portion of the flagellum, which can be correlated with IFT172 antibody labeling studies .

• Live imaging of fluorescently tagged IFT172: While not directly using antibodies, this approach can complement antibody-based studies to investigate the dynamics of IFT172 in living cells.

Why might I observe inconsistent IFT172 staining patterns in ciliated cells?

Inconsistent staining patterns with IFT172 antibody can result from several methodological factors:

• Developmental stage variations: IFT172 distribution changes during ciliogenesis. In the proximal portion of the flagellum, IFT proteins initially form a ring surrounding all 9 doublet microtubules (DMTs), but later become restricted to specific DMTs (3-4 and 7-8 in Trypanosoma brucei) .

• Fixation-dependent epitope masking: The complex structure of IFT172, including its U-box domain and TPR motifs, can be sensitive to different fixation methods. For IHC applications with formalin-fixed tissues, appropriate antigen retrieval is crucial - use TE buffer at pH 9.0 or alternatively citrate buffer at pH 6.0 .

• Antibody specificity issues: Ensure the antibody recognizes the appropriate epitope. The antibody described in the results targets a fusion protein antigen (Ag28921) , so its recognition may depend on protein conformation.

• Cell type-specific expression patterns: IFT172 expression varies across tissues and cell types. Validated positive samples include mouse testis tissue, rat testis tissue, mouse brain tissue, and ARPE-19 cells .

• Protein degradation during sample preparation: IFT172 is a large protein (198 kDa calculated, 180 kDa observed) that may be susceptible to proteolysis. Use fresh samples and include protease inhibitors throughout preparation.

How can I optimize IFT172 detection in cells with low expression levels?

To enhance detection sensitivity for low-abundance IFT172, implement these evidence-based optimization strategies:

• Signal amplification systems: Consider using tyramide signal amplification (TSA) or other enzymatic amplification methods to boost fluorescence signal while maintaining specificity.

• Antibody concentration optimization: For samples with low expression, adjust antibody concentration to the higher end of the recommended dilution range (closer to 1:50 for IF/ICC or IHC) .

• Extended primary antibody incubation: Increase incubation time (overnight at 4°C) to enhance binding to low-abundance targets while maintaining low background.

• Sample enrichment techniques: If studying ciliary localization, consider methods to enrich for ciliated cells or isolate cilia to concentrate the target protein.

• Blocking optimization: Test different blocking reagents (BSA, normal serum, commercial blockers) to reduce background while maximizing specific signal.

• Detector sensitivity settings: When using digital imaging systems, optimize exposure settings to capture low-level signals without introducing artifacts.

What controls should be included when using IFT172 antibody in colocalization studies?

For rigorous IFT172 colocalization experiments, incorporate these methodological controls:

• Single-antibody controls: Process samples with each antibody individually to establish baseline staining patterns and verify that bleed-through between channels does not occur.

• Isotype controls: Include appropriate isotype controls (Rabbit IgG for the 28441-1-AP antibody) to assess non-specific binding.

• Known interaction partners: Include positive control antibodies against established IFT172 interaction partners, such as IFT144 and IFT140 for C-terminal interactions or IFT57/IFT80 for N-terminal interactions .

• Non-ciliated cell controls: Include non-ciliated cells or areas without cilia as negative controls for ciliary staining specificity.

• Antibody cross-reactivity assessment: When using antibodies from different species, include controls to verify that secondary antibodies do not cross-react.

• Quantitative colocalization metrics: Apply appropriate statistical measures (Pearson's correlation coefficient, Manders' overlap coefficient) to quantify colocalization rather than relying solely on visual assessment.

How can super-resolution microscopy enhance IFT172 antibody-based studies?

Super-resolution microscopy offers significant advantages for IFT172 research through these methodological applications:

• Nanoscale localization precision: Super-resolution techniques provide approximately 20-50 nm resolution, enabling visualization of IFT172 distribution along specific doublet microtubules within the axoneme, which cannot be resolved with conventional microscopy .

• Multi-protein complex architecture: Super-resolution microscopy can reveal the spatial organization of IFT172 within the IFT complex, particularly its bridging role between IFT-A and IFT-B subcomplexes .

• Dynamic processes visualization: Techniques like STORM have demonstrated that IFT proteins initially associate with all nine DMTs in the proximal flagellum before becoming restricted to specific doublets (DMTs 3-4 and 7-8) , revealing dynamic processes impossible to observe with conventional microscopy.

• Quantitative distribution analysis: Super-resolution data enables precise quantification of IFT172 protein distribution along ciliary structures and comparison between normal and mutant conditions.

• Correlative approaches: Combining super-resolution with expansion microscopy (U-ExM) provides both improved resolution and molecular identification capabilities, as demonstrated in studies of IFT distribution in Trypanosoma brucei .

What strategies can be employed to study post-translational modifications of IFT172?

To investigate post-translational modifications (PTMs) of IFT172, apply these specialized methodological approaches:

• Ubiquitination analysis: Use anti-ubiquitin antibodies in combination with IFT172 antibodies to detect mono-ubiquitination and potential poly-ubiquitination. IFT172 has demonstrated auto-ubiquitination activity in vitro, particularly generating a strong mono-ubiquitinated band .

• Phosphorylation studies: Employ phospho-specific antibodies or Phos-tag gels to detect phosphorylation events that might regulate IFT172 function or complex assembly.

• U-box domain mutant analysis: The U-box domain contains key residues (I1688, F1715, P1725) important for ubiquitination activity . Generate mutations in these residues and use antibodies to assess both IFT172 expression levels and ubiquitination patterns.

• Mass spectrometry validation: Following immunoprecipitation with IFT172 antibodies, use mass spectrometry to identify and map specific PTM sites, which can then be targeted for functional studies.

• PTM-dependent interaction studies: Use IFT172 antibodies in pulldown experiments under conditions that preserve or remove specific PTMs to determine how modifications affect protein-protein interactions, particularly with IFT-A components (IFT144, IFT140).

How can IFT172 antibodies contribute to understanding ciliopathy disease mechanisms?

IFT172 antibodies provide critical tools for elucidating ciliopathy disease mechanisms through:

• Patient variant functional analysis: Multiple missense variants from ciliopathy patients map to interfaces in IFT172's structure . Use antibodies to assess how these mutations affect protein expression, localization, and function in patient-derived or engineered cells.

• Protein stability assessment: In cells with U-box deletion in IFT172, immunostaining reveals striking reduction in protein levels, suggesting the U-box domain is crucial for protein stability . Antibodies enable quantitative measurement of these effects.

• Retrograde IFT defect characterization: The FLA11 mutation in IFT172's TPR motif directly impacts association with IFT-A subunits, causing retrograde IFT defects characterized by accumulation of IFT proteins at the ciliary tip . Antibody staining can visualize this phenotype.

• Compensatory mechanism identification: In heterozygous IFT172ΔU-box cells, upregulation of the wild-type allele compensates for reduced expression of the mutant allele . Antibodies allow detection of these compensatory responses.

• Pathway analysis: Antibodies against IFT172 and other ciliary proteins can be used to investigate signaling pathways affected in ciliopathies, such as Hedgehog signaling, which relies on proper cilia function.

What considerations are important when using IFT172 antibodies for electron microscopy studies?

When employing IFT172 antibodies for electron microscopy applications, address these methodological considerations:

• Epitope preservation: Conventional EM fixation and embedding procedures can destroy antibody epitopes. Consider using specialized techniques like Tokuyasu cryosectioning or high-pressure freezing and freeze substitution to better preserve antigenicity.

• Antibody penetration: For post-embedding immunogold labeling, use ultrathin sections (70-90nm) and appropriate etching or antigen retrieval steps to improve antibody access to epitopes.

• Correlation with fluorescence data: Use correlative light and electron microscopy (CLEM) to directly relate IFT172 immunofluorescence patterns with ultrastructural features observed in EM.

• Immunogold labeling optimization: For optimal signal-to-noise ratio, determine the appropriate gold particle size and antibody dilution through systematic testing. Secondary antibodies conjugated to 5-10nm gold particles typically provide good resolution for intracellular structures.

• Structural context interpretation: Volume electron microscopy has revealed IFT-like densities along microtubule doublets in the proximal portion of the flagellum . Immunogold labeling can confirm these structures contain IFT172 and contribute to understanding how IFT trains assemble.

How can genetic modification approaches be combined with IFT172 antibody studies?

Integrate genetic engineering with antibody-based detection through these methodological strategies:

• CRISPR-Cas9 gene editing: Generate IFT172 knockout or knock-in cell lines (such as the IFT172ΔU-box cells described in the literature) and use antibodies to validate modification efficiency and study resulting phenotypes.

• Domain-specific mutations: Introduce mutations in specific domains (TPR motifs or U-box domain) based on structural information and use antibodies to assess effects on protein expression, localization, and function.

• Patient variant modeling: Recreate ciliopathy-associated mutations like C1727R in cell or animal models and use antibodies to characterize their effects compared to wild-type IFT172.

• Rescue experiments: In IFT172-depleted backgrounds, express wild-type or mutant versions of IFT172 and use antibodies to assess restoration of normal protein levels and localization.

• Inducible expression systems: Create cell lines with inducible IFT172 expression or depletion and use antibodies to monitor dynamic changes in protein levels and localization during ciliogenesis.