IFT172 Antibody, FITC conjugated
Description
Fundamental Characteristics of IFT172 Antibody, FITC Conjugated
IFT172 Antibody, FITC conjugated is an immunological reagent specifically designed to detect and visualize the Intraflagellar Transport 172 protein. The antibody is produced by immunizing rabbits with synthetic peptides derived from human IFT172 protein, followed by purification and conjugation with FITC, a commonly used fluorescent dye with excitation and emission wavelengths of approximately 495 nm and 519 nm, respectively. The FITC conjugation eliminates the need for secondary antibody labeling, allowing for direct detection of the target protein in fluorescence-based applications .
Commercial preparations of IFT172 antibody with FITC conjugation are available from several manufacturers, with varying specifications regarding the epitope region, host species, and validation parameters. For instance, Bioss offers a polyclonal IFT172 antibody (catalog #bs-15560R-FITC) derived from KLH-conjugated synthetic peptide from the 1-100/1749 amino acid range of human IFT172 . Other variants include antibodies targeting specific amino acid regions such as 1368-1502, as seen in the ABIN7156975 product .
The antibody's polyclonal nature provides advantages in terms of epitope recognition, as it can bind to multiple antigenic determinants on the target protein, potentially enhancing detection sensitivity across various experimental conditions and species.
Molecular and Functional Properties of IFT172 Protein
IFT172 is a critical component of the intraflagellar transport machinery, a bidirectional movement system essential for the assembly, maintenance, and function of cilia and flagella. Understanding the protein's properties is crucial for appropriate application of the corresponding antibody in research contexts.
IFT172 functions as a membrane-associated protein that directly interacts with and remodels the cell membrane. Structurally, purified IFT172 exhibits two spherical domains with a long rod protrusion, referred to as an open conformation. This architecture bears similarity to proteins like clathrin or COPI-II, which are involved in vesicular transport . The protein's ability to interact with lipids is mediated through the β-helical region of its N-terminal globular domain, which can bind to lipid membranes and remodel them into small vesicles approximately 20 nm in size .
Within the intraflagellar transport system, IFT172 serves as a modulatory cargo adapter that initially anchors at the primary ciliary base before recruiting other IFT components to complete assembly . Additionally, research has demonstrated that IFT172 interacts with IFT57, a component of IFT-B2, through a competitive binding mechanism that prevents IFT complex degradation .
Validated Applications and Methodological Considerations
IFT172 antibody, FITC conjugated has been validated for several immunological applications, with particular utility in fluorescence-based detection methods. The direct FITC conjugation eliminates the need for secondary antibody incubation, potentially reducing background and cross-reactivity while streamlining experimental protocols.
Application Range and Dilution Recommendations
The primary applications for IFT172 antibody, FITC conjugated include:
| Application | Recommended Dilution |
|---|---|
| Immunofluorescence (IHC-P) | 1:50-200 |
| Immunofluorescence (IHC-F) | 1:50-200 |
| Immunofluorescence (ICC) | 1:50-200 |
These application parameters provide researchers with starting points for optimization in their specific experimental systems .
Immunofluorescence Protocol Considerations
When using IFT172 antibody, FITC conjugated for immunofluorescence applications, researchers typically follow protocols that involve:
-
Sample preparation through fixation with 4% paraformaldehyde (10 minutes) or methanol (5 minutes at -20°C)
-
Permeabilization with 0.1% Nonidet P-40 for PFA-fixed samples
-
Blocking with PBS containing 0.1% bovine serum albumin for 45-60 minutes
-
Incubation with diluted antibody for 45-60 minutes
-
Washing steps to remove unbound antibody
-
DAPI staining for visualization of nuclear DNA
-
Mounting and observation with appropriate fluorescence microscopy equipment
The FITC conjugation allows for direct visualization in the green fluorescence channel without the need for secondary antibody incubation, simplifying the workflow compared to unconjugated primary antibodies.
Research Applications in Ciliary Biology and Disease Models
IFT172 antibody, FITC conjugated serves as a valuable tool in investigating various aspects of ciliary biology and associated pathological conditions. The protein's critical role in intraflagellar transport makes it an important marker for studying ciliopathies—congenital diseases resulting from dysfunctional primary cilia.
Ciliopathies present with multifaceted clinical manifestations including retinopathy, congenital kidney disease, intellectual disability, and syndromes such as Bardet-Biedl syndrome . By enabling direct visualization of IFT172 localization and dynamics, FITC-conjugated antibodies facilitate research into the molecular mechanisms underlying these conditions.
In model organisms such as Trypanosoma brucei, IFT172 antibodies have been employed to study the intriguing process of simultaneous maintenance of existing flagella while assembling new ones. Immunostaining with anti-IFT172 has revealed the presence of IFT proteins along the axoneme and at the basal body and probasal body regions of both old and new flagella . Such studies provide insights into the conservation of intraflagellar transport mechanisms across species and their functional significance.
Electron microscopy studies complemented by immunofluorescence have demonstrated that IFT particles exhibit specific localization patterns, suggesting the existence of dedicated IFT tracks along axonemal microtubules. This spatial organization appears crucial for the bidirectional movement of IFT complexes and the proteins they transport .
Comparative Analysis with Other IFT172 Antibody Formats
To fully appreciate the utility of FITC-conjugated IFT172 antibodies, it is valuable to compare them with other available formats. Various manufacturers offer IFT172 antibodies with different conjugations or in unconjugated forms, each with specific advantages depending on the research application.
| Antibody Format | Catalog Example | Key Advantages | Primary Applications |
|---|---|---|---|
| FITC Conjugated | bs-15560R-FITC | Direct detection, no secondary antibody needed | IF(IHC-P), IF(IHC-F), IF(ICC) |
| Unconjugated | 28441-1-AP | Flexibility with secondary antibody selection | WB, IHC, IF/ICC, ELISA |
| HRP Conjugated | Various | Direct detection in enzymatic assays | ELISA, WB |
| Biotin Conjugated | Various | Amplification via avidin-biotin system | ELISA, IHC |
The selection between these formats depends on specific research requirements, including the desired detection sensitivity, availability of imaging equipment, and experimental design considerations .
The unconjugated antibody formats offer greater flexibility in detection strategies but require additional incubation steps with secondary antibodies. In contrast, directly conjugated antibodies like the FITC variant streamline protocols but may have predetermined detection parameters based on the fluorophore properties.
Practical Considerations for Optimal Results
Achieving optimal results with IFT172 antibody, FITC conjugated requires attention to several practical considerations throughout the experimental workflow. Storage conditions significantly impact antibody performance, with recommendations to store the reagent at -20°C and aliquot it to avoid repeated freeze-thaw cycles that could compromise activity .
Dilution optimization is another critical factor, with manufacturers providing recommended ranges rather than definitive values. Researchers are advised to conduct titration experiments to determine the optimal antibody concentration for their specific experimental system, balancing signal intensity against background fluorescence .
When analyzing results, it is important to include appropriate controls, such as samples where the primary antibody is omitted or replaced with non-specific IgG from the same host species. These controls help distinguish specific IFT172 staining from potential autofluorescence or non-specific binding artifacts.
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- This is the second reported instance of IFT172 mutations in Bardet-Biedl syndrome patients, confirming IFT172 as the twentieth BBS gene (BBS20). PMID: 26763875
- Research has identified mutations in IFT172 that lead to a recessive form of non-syndromic retinitis pigmentosa and Bardet-Biedl syndrome. These findings suggest that primary IFT172 mutations alone may not fully explain the broad range of phenotypes observed. PMID: 25168386
- Defects in IFT172 have been identified as a cause of complex asphyxiating thoracic dystrophy and Mainzer-Saldino syndrome. PMID: 24140113
Q&A
What is IFT172 and why is it important in ciliary research?
IFT172 is a key component of the intraflagellar transport (IFT) machinery, essential for the assembly and maintenance of cilia and flagella. It serves as a critical bridge between IFT-B and IFT-A complexes in both anterograde and retrograde IFT trains, facilitating bidirectional transport along the cilium/flagellum . The protein contains functionally relevant motifs at its C-terminus: a TPR motif involved in IFT-A association and a U-box-like domain likely involved in ciliary ubiquitination events . IFT172's significance stems from its dual functionality in providing structural support for IFT and regulating signaling pathways, explaining why mutations in this protein lead to diverse ciliopathy phenotypes .
What fixation methods are optimal for IFT172 immunofluorescence studies?
Based on published protocols, two effective fixation methods for IFT172 immunofluorescence have been established:
-
Paraformaldehyde (PFA) fixation: Cells should be fixed in 4% PFA for 10 minutes, followed by permeabilization with 0.1% Nonidet P-40 for 10 minutes .
-
Methanol fixation: Cells can be fixed in methanol for a maximum of 5 minutes at -20°C .
Following fixation, samples should be blocked in PBS containing 0.1% bovine serum albumin for 45-60 minutes, then incubated with the primary antibody at a recommended dilution of 1:200 for 45-60 minutes . For non-conjugated antibodies, subsequent incubation with appropriate secondary antibodies is required, whereas FITC-conjugated IFT172 antibodies eliminate this need.
How can I optimize the detection of IFT172 at the ciliary base versus the ciliary tip?
Detecting IFT172 at specific ciliary locations requires careful consideration of fixation methods and imaging techniques. Recent cryo-ET structures have revealed that IFT172's C-terminus interacts with IFT144 in anterograde trains and with IFT140 in retrograde trains . This differential association suggests location-specific conformational changes in IFT172.
Methodological approach:
-
Use detergent-extracted cytoskeletons for improved visualization of ciliary structures
-
Apply super-resolution microscopy techniques (such as STED or STORM) to distinguish between ciliary base and tip localization
-
Consider dual-labeling approaches with markers for the basal body (e.g., gamma-tubulin) and ciliary tip (e.g., EB1)
-
Compare methanol versus PFA fixation, as each may differentially preserve IFT172 epitopes based on protein conformation at different ciliary locations
-
Optimize antibody concentration and incubation times based on subcellular localization interest (typically higher concentrations may be needed for tip detection)
How does IFT172's U-box domain influence experimental design for studying ubiquitination in cilia?
The discovery that IFT172 contains a U-box domain with potential ubiquitin ligase activity opens new avenues for investigating ubiquitination events in cilia. When designing experiments to study this function, researchers should consider:
Experimental approaches:
-
Co-immunoprecipitation studies using FITC-conjugated IFT172 antibodies to capture and identify ubiquitinated targets
-
In vitro ubiquitination assays to assess IFT172's ligase activity, using purified components including:
-
Mutational analysis of the IFT172 U-box domain to identify residues critical for ubiquitin binding and potential ligase activity
-
Analysis of ubiquitination patterns in wild-type versus IFT172 U-box mutant cells
Data from in vitro ubiquitination studies of IFT172:
What are the critical parameters for successful immunolabeling of IFT172 in different model organisms?
When working with IFT172 antibodies across various model organisms, researchers must consider evolutionary conservation and potential cross-reactivity. IFT172 is highly conserved from unicellular eukaryotes to humans , but epitope accessibility may vary.
Key methodological considerations:
-
Chlamydomonas reinhardtii:
-
Tetrahymena:
-
Mammalian cells:
How can I quantitatively assess IFT172 dynamics in live cells using FITC-conjugated antibodies?
For live-cell imaging studies, FITC-conjugated IFT172 antibodies must be carefully introduced into cells while maintaining cell viability and minimizing functional disruption.
Recommended protocol:
-
Antibody delivery options:
-
Microinjection of FITC-conjugated antibodies (1-5 ng/μL)
-
Cell-penetrating peptide conjugation to facilitate cellular uptake
-
Electroporation under gentle conditions (typically 250V, 950μF for mammalian cells)
-
-
Imaging parameters:
-
Use low-phototoxicity conditions (reduced laser power, 5-10% of maximum)
-
Implement resonant scanning or spinning disk confocal microscopy for rapid acquisition
-
Consider photobleaching correction algorithms in analysis
-
-
Quantitative analysis approach:
-
Track individual IFT particles using particle tracking software (e.g., TrackMate in ImageJ)
-
Measure velocity, direction, and frequency of IFT172-positive particles
-
Calculate particle flux rates at ciliary base and tip
-
Apply kymograph analysis to visualize bidirectional transport dynamics
-
How should I interpret contradictory results between fixed-cell and live-cell imaging of IFT172?
Researchers frequently encounter discrepancies between fixed and live-cell observations when studying IFT172. These differences often arise from:
-
Fixation artifacts: Methanol and PFA fixation can differentially preserve IFT172 conformations or interactions
-
Temporal resolution limitations: Fixed samples capture a single time point, while IFT is a highly dynamic process
-
Epitope masking: IFT172's interactions with IFT-A components (IFT144, IFT140) may mask antibody binding sites in certain transport states
Recommended approaches to reconcile contradictory data:
-
Validate findings using multiple fixation methods
-
Complement antibody studies with fluorescently tagged IFT172 constructs
-
Consider the specific IFT172 domain being recognized by the antibody (N-terminal vs C-terminal)
-
Integrate fixed and live imaging data using computational modeling approaches
-
Assess potential artifacts through careful controls and comparative analysis across methodologies
What strategies can resolve non-specific background in IFT172-FITC antibody experiments?
High background is a common challenge with FITC-conjugated antibodies due to tissue autofluorescence in the green spectrum and potential non-specific binding.
Troubleshooting strategies:
-
Optimize blocking conditions:
-
Extend blocking time to 1-2 hours
-
Test alternative blocking agents (normal serum, casein, commercial blocking buffers)
-
Include 0.1-0.3% Triton X-100 in blocking buffer for improved permeabilization
-
-
Reduce autofluorescence:
-
Treat samples with sodium borohydride (0.1% for 15 minutes) prior to blocking
-
Use Sudan Black B (0.1% in 70% ethanol) post-staining
-
Implement spectral unmixing during image acquisition
-
-
Antibody optimization:
-
Titrate antibody concentration to determine optimal signal-to-noise ratio
-
Include negative controls using non-ciliated cells or IFT172-knockout samples
-
Consider using Fab fragments for reduced non-specific binding
-
How can IFT172-FITC antibodies be employed to investigate the relationship between IFT and ciliary signaling pathways?
Recent findings highlight IFT172's involvement in regulating signaling pathways, particularly TGF-beta/BMP signaling . FITC-conjugated IFT172 antibodies can be leveraged for investigating these connections through:
Methodological approaches:
-
Co-localization studies:
-
Dual immunolabeling of IFT172-FITC and signaling components (receptors, effectors)
-
Super-resolution microscopy to resolve spatial relationships at nanometer scale
-
Time-course experiments to track temporal dynamics during signaling activation
-
-
Proximity ligation assays (PLA):
-
Combine IFT172-FITC antibodies with antibodies against signaling proteins
-
Quantify interaction events at different ciliary regions and time points
-
Compare PLA signals between wild-type and signaling pathway mutants
-
-
FRET-based approaches:
-
Pair IFT172-FITC antibodies with acceptor fluorophore-conjugated signaling component antibodies
-
Measure energy transfer efficiency as indicator of molecular proximity
-
Map interaction domains through selective mutagenesis
-
What is the significance of IFT172's dual interaction with IFT144 and IFT140 for experimental design?
The discovery that IFT172's C-terminus can interact with either IFT144 (in anterograde transport) or IFT140 (in retrograde transport) through mutually exclusive binding has important implications for experimental design:
Experimental considerations:
-
Mutational analysis strategies:
-
Target the conserved TPR motif implicated in both interactions
-
Generate selective mutations that disrupt only one interaction while preserving the other
-
Assess the functional consequences on anterograde versus retrograde transport
-
-
Structural studies approach:
-
Functional assays:
-
Compare ciliary phenotypes between mutations affecting IFT144 binding versus IFT140 binding
-
Analyze the consequences for IFT train assembly and dynamics
-
Investigate potential regulatory mechanisms governing the switch between binding partners
-
