IFT172 Antibody, Biotin conjugated

What is IFT172 and why is it important in cellular research?

IFT172 is the largest protein of the Intraflagellar Transport (IFT) complex, playing a crucial role in cilium formation across eukaryotic organisms. It functions as a bridge between IFT-A and IFT-B complexes, facilitating proper ciliary assembly and function . Recent structural studies have revealed that IFT172 contains a conserved U-box-like domain often found in E3 ubiquitin ligases, exhibiting ubiquitin-binding properties and auto-ubiquitination activity . This dual functionality—providing both structural support and signaling regulation—explains why IFT172 mutations lead to diverse ciliopathy phenotypes. Studying IFT172 is essential for understanding ciliary biology, development, and disease mechanisms .

What are the key specifications of the IFT172 Antibody, Biotin conjugated?

The IFT172 Antibody, Biotin conjugated (Product Code: CSB-PA887019LD01HU) is a polyclonal antibody raised in rabbits against recombinant Human Intraflagellar transport protein 172 homolog protein (amino acids 1368-1502) . It demonstrates reactivity with human IFT172 (UniProt: Q9UG01) and has been tested for ELISA applications. The antibody is supplied in liquid form containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative . It has been purified to >95% purity using Protein G purification methods and is conjugated with biotin for enhanced detection capability . This preparation allows for sensitive detection of IFT172 in various experimental contexts.

How does the biotin conjugation enhance the utility of the IFT172 antibody in research?

Biotin conjugation significantly enhances the IFT172 antibody's utility by enabling amplified signal detection through the strong biotin-avidin/streptavidin interaction system. This modification allows researchers to implement multi-layered detection systems where streptavidin-conjugated reporter molecules (enzymes, fluorophores) bind specifically to the biotinylated antibody, increasing sensitivity without compromising specificity . The biotin conjugation provides flexibility in detection methods, allowing the same antibody to be used across various experimental platforms including ELISA, immunohistochemistry, and flow cytometry. Additionally, this conjugation reduces the need for secondary antibodies in many applications, simplifying experimental protocols and potentially reducing background signal in complex biological samples where IFT172 localization and function are being investigated .

What are the optimal storage and handling conditions for maintaining IFT172 Antibody activity?

For optimal maintenance of IFT172 Antibody activity, store the antibody at -20°C or -80°C immediately upon receipt . Critically, avoid repeated freeze-thaw cycles as these can progressively degrade antibody structure and compromise biotin conjugation integrity. When handling the antibody, maintain sterile conditions and use nuclease-free equipment to prevent contamination. For routine experimental work, prepare small working aliquots (20-50 μL) in sterile microcentrifuge tubes to minimize freeze-thaw events . Before each use, thaw aliquots completely at 4°C (not room temperature) and mix gently by finger-tapping or slow pipetting rather than vortexing, which can damage antibody structure. The antibody formulation contains 50% glycerol, which helps stabilize protein structure during freeze-thaw transitions, but does not eliminate degradation risks entirely . For experiments requiring diluted antibody solutions, prepare fresh dilutions immediately before use as biotin-conjugated antibodies may show reduced stability in dilute solutions.

How should appropriate dilutions for IFT172 Antibody be determined for ELISA applications?

Determining appropriate dilutions for IFT172 Antibody in ELISA applications requires systematic optimization. Begin with a preliminary titration experiment using broad dilution ranges (1:100, 1:500, 1:1000, 1:5000, and 1:10000) against purified recombinant IFT172 protein at a fixed concentration (typically 1-5 μg/mL) . The ideal working dilution should yield signal-to-noise ratios of >10:1 between positive samples and negative controls. For more precise optimization, conduct a secondary titration within the narrower range identified in the preliminary test, using both target samples and appropriate controls. Since this antibody recognizes amino acids 1368-1502 of human IFT172 protein, ensure your recombinant proteins or test samples contain this epitope region . When working with complex biological samples such as cell lysates, include additional controls such as IFT172-knockout samples to verify specificity. Document batch-to-batch variations by maintaining detailed records of optimal dilutions for each antibody lot, as minor manufacturing differences can affect required concentrations .

What methods can be used to validate the specificity of IFT172 Antibody in experimental systems?

Validating IFT172 Antibody specificity requires multiple complementary approaches. First, perform western blotting with cell lysates from both IFT172-expressing and IFT172-knockout/knockdown models, confirming a single band at the expected molecular weight (~172 kDa) that disappears in knockout samples . Second, conduct immunoprecipitation followed by mass spectrometry to verify that IFT172 is the predominant protein captured by the antibody. Third, implement immunofluorescence studies in ciliated cells, where proper IFT172 localization should show enrichment at the ciliary base and tip in accordance with its established distribution pattern . For heterologous expression systems, compare antibody signals between cells expressing tagged IFT172 constructs (full-length vs. N-terminal only vs. C-terminal only) to confirm epitope recognition, particularly important since this antibody targets the C-terminal region (amino acids 1368-1502) . Additionally, peptide competition assays using the immunogen peptide sequence should abolish specific staining if the antibody is truly specific. Cross-reactivity testing against related proteins, particularly IFT57 and IFT80 which interact with IFT172, should show minimal recognition .

How can IFT172 Antibody be used to investigate the structural bridge between IFT-A and IFT-B complexes?

The IFT172 Antibody provides a powerful tool for investigating the structural bridge between IFT-A and IFT-B complexes through several sophisticated approaches. Implement proximity-based interactome mapping by combining immunoprecipitation of IFT172 with subsequent mass spectrometry to identify associated proteins from both IFT-A (particularly IFT144 and IFT140) and IFT-B complexes (IFT57/IFT80) . Since the antibody targets amino acids 1368-1502, it recognizes regions near the C-terminal TPR motif involved in IFT-A association, making it particularly valuable for examining these interactions . For higher spatial resolution, employ super-resolution microscopy techniques (STORM, PALM) using fluorophore-conjugated streptavidin to visualize the biotinylated IFT172 antibody in relationship to other IFT complex components. The bridging function can be directly tested through in vitro reconstitution experiments, where purified IFT-A and IFT-B subcomplexes are mixed with and without IFT172, and complex formation is monitored using the biotinylated antibody as a detection reagent . Additionally, utilize cryo-electron tomography of isolated IFT trains from ciliated cells labeled with gold-conjugated streptavidin bound to the biotinylated antibody to precisely localize IFT172 within the three-dimensional architecture of IFT trains .

What approaches can be used to study the newly discovered U-box domain and ubiquitination activity of IFT172?

To study the U-box domain and ubiquitination activity of IFT172, researchers should implement a multi-faceted experimental strategy. Begin with in vitro ubiquitination assays using purified IFT172 C-terminal fragments (containing the U-box domain), E1/E2 enzymes, ubiquitin, and ATP, followed by Western blotting with the IFT172 Antibody to detect self-ubiquitination . Perform E2 enzyme screening assays to identify specific E2 partners, focusing particularly on the UbcH5 E2 enzyme family which has shown activity with IFT172 . For structure-function analyses, generate point mutations in conserved U-box residues (I1688, F1715, P1725) and the disease-associated C1727R variant, then assess how these mutations affect ubiquitination activity . The biotin-conjugated IFT172 antibody can be particularly valuable in pull-down assays to identify potential ubiquitination substrates in ciliary extracts. To investigate the ubiquitin-binding properties, implement pulldown assays with tetra-ubiquitin chains and GST-tagged IFT172 C-terminal fragments, using the antibody for detection . For cellular studies, perform co-localization analysis of ubiquitinated proteins and IFT172 within cilia using dual-labeling approaches. Finally, investigate the physiological significance by examining ciliary protein trafficking in cells expressing wild-type versus U-box-mutant IFT172, monitoring changes in ubiquitination patterns of ciliary proteins .

How can IFT172 Antibody contribute to understanding ciliopathy disease mechanisms?

The IFT172 Antibody can significantly advance understanding of ciliopathy disease mechanisms through multiple research approaches. Implement comparative immunostaining in patient-derived cells harboring IFT172 mutations (especially D1605E and C1727R variants) versus control cells to assess changes in IFT172 localization, abundance, and distribution within cilia . The biotin conjugation enables sensitive detection of potentially reduced IFT172 levels in patient samples. Develop proximity labeling assays where biotinylated IFT172 antibody is used to identify differential protein interactions between wild-type and mutant IFT172 in cellular contexts. For functional studies, examine how patient mutations affect the bridging capacity of IFT172 between IFT-A and IFT-B complexes by comparing co-immunoprecipitation efficiency in cells expressing wild-type versus mutant IFT172 . Investigate the impact on signaling pathways, particularly TGFB signaling which has been linked to IFT172 function, by assessing receptor trafficking and processing within cilia in disease models . For in vivo relevance, create animal models expressing specific human ciliopathy variants and use the antibody to track IFT172 distribution in affected tissues. Additionally, evaluate how mutations in the U-box domain (particularly C1727R) affect ubiquitination activity and subsequent ciliary protein homeostasis, providing mechanistic insights into how disrupted ubiquitin-related activities contribute to disease manifestation .

What are common challenges when using IFT172 Antibody in immunofluorescence, and how can they be overcome?

Common challenges with IFT172 Antibody in immunofluorescence include insufficient signal intensity, high background, and non-specific staining. To overcome these issues, implement a systematic optimization approach. For insufficient signal, employ a streptavidin-based signal amplification system (using fluorophore-conjugated streptavidin) to enhance the detection of biotinylated antibody . Optimize fixation methods—4% paraformaldehyde typically preserves IFT172 epitopes while maintaining ciliary structure, but methanol fixation may better expose the C-terminal epitope (amino acids 1368-1502) that this antibody recognizes . When encountering high background, increase blocking stringency by using 5-10% normal serum from the same species as secondary reagents plus 0.1-0.3% Triton X-100, and consider adding 0.1% BSA to all wash buffers. For non-specific staining, pre-absorb the antibody with acetone powder from non-relevant tissues or implement antigen competition controls with the immunizing peptide (amino acids 1368-1502) . Ciliary visualization can be challenging due to their small size—combine the IFT172 staining with established ciliary markers like acetylated tubulin and employ super-resolution microscopy techniques to resolve subciliary localization . For samples with low IFT172 expression, consider tyramide signal amplification methods compatible with biotin-conjugated primary antibodies.

How can quantitative analysis of IFT172 levels be optimized using this antibody in Western blotting?

Optimizing quantitative analysis of IFT172 levels in Western blotting requires attention to several critical factors. First, establish a standardized protein extraction protocol that effectively solubilizes IFT172 from ciliary and basal body compartments, typically requiring stronger lysis buffers containing 1% NP-40 or Triton X-100 with brief sonication . For SDS-PAGE, use 6-8% gels to properly resolve the large IFT172 protein (~172 kDa), and transfer to PVDF membranes (rather than nitrocellulose) at lower amperage for extended periods (overnight at 30V) to ensure complete transfer of high molecular weight proteins . When detecting with streptavidin-HRP, implement a concentration-dependent standard curve using recombinant IFT172 protein spanning the epitope region (amino acids 1368-1502) to establish a linear detection range. To minimize lane-to-lane variation, normalize IFT172 signals to multiple housekeeping proteins using multiplexed fluorescent detection systems . For quantifying IFT172 in different subcellular fractions, develop fractionation protocols that separate ciliary, basal body, and cytoplasmic compartments with minimal cross-contamination. Validate antibody specificity in each experimental system by including IFT172-knockout/knockdown controls, confirming absence of signal in these samples . When comparing IFT172 levels across multiple conditions, process all samples in parallel using identical reagent lots and exposure parameters to minimize technical variability.

What strategies can improve IFT172 detection in limited or challenging samples?

For improved IFT172 detection in limited or challenging samples, implement multiple signal enhancement strategies tailored to biotin-conjugated antibodies. When working with minimal ciliated cell populations, employ microfluidic cell isolation techniques followed by whole-cell immunostaining to increase starting material purity before analysis . For formalin-fixed paraffin-embedded tissues where epitope masking often occurs, optimize antigen retrieval methods specifically for IFT172, testing both heat-induced epitope retrieval (HIER) with citrate buffer (pH 6.0) and Tris-EDTA buffer (pH 9.0) to identify optimal conditions . In highly autofluorescent samples, utilize spectral imaging coupled with linear unmixing algorithms to separate specific IFT172 signal from background. For ultra-sensitive detection in minimal samples, implement rolling circle amplification (RCA) methods, where streptavidin-conjugated DNA primers bind to biotinylated antibodies, enabling exponential signal amplification through DNA polymerization . In co-localization studies involving multiple antibodies, use primary antibodies from different host species to prevent cross-reactivity, or employ sequential staining protocols with appropriate blocking steps between antibody applications. For single-cell analysis of IFT172 distribution, combine the antibody detection with expansion microscopy techniques, physically enlarging samples to reveal subciliary details normally below the diffraction limit .

How might the IFT172 Antibody be used to investigate the relationship between cilia and the TGFB signaling pathway?

The IFT172 Antibody presents unique opportunities for investigating the relationship between cilia and TGFB signaling through several innovative approaches. Researchers can develop co-immunoprecipitation protocols using the biotinylated antibody to pull down IFT172-associated protein complexes from ciliated cells under various TGFB stimulation conditions, identifying dynamic interactome changes during signal transduction . The antibody enables high-resolution co-localization studies between IFT172 and TGFB pathway components (receptors, Smad proteins) using super-resolution microscopy techniques, potentially revealing novel spatial relationships within the ciliary compartment . For functional studies, combine TGFB pathway manipulation (stimulation, inhibition) with real-time tracking of IFT172-positive particles within cilia using live-cell imaging approaches based on fluorescently-tagged streptavidin binding to the biotinylated antibody. Investigate whether IFT172's U-box domain directly ubiquitinates TGFB receptors by implementing in vitro ubiquitination assays with purified components, followed by mass spectrometry to identify modification sites . For disease relevance, compare TGFB signaling responses in cells expressing wild-type versus U-box-mutant IFT172 (especially the C1727R variant), examining receptor trafficking, internalization rates, and downstream signaling activation. The antibody also enables precise quantification of IFT172 association with TGFB receptors at different ciliary subdomains (base, shaft, tip) under various signaling conditions .

What novel approaches could combine this antibody with emerging technologies to study IFT172 dynamics in live cells?

Combining the IFT172 Antibody with emerging technologies offers revolutionary approaches to studying IFT172 dynamics in live cells. Develop antibody internalization protocols using cell-permeable streptavidin conjugates that can bind to biotinylated IFT172 antibody fragments introduced into cells via microinjection or protein transfection . For high temporal resolution imaging, couple the antibody with quantum dots or photostable organic fluorophores linked to streptavidin, enabling long-term single-particle tracking of IFT172-containing complexes within living cilia . Implement optogenetic approaches where light-activated streptavidin variants bind to biotinylated antibodies at specific timepoints, allowing temporal control over IFT172 complex manipulation. For quantitative biophysical measurements, combine the antibody with fluorescence correlation spectroscopy (FCS) to measure diffusion coefficients and complex formation dynamics of IFT172 at different ciliary regions . Develop CRISPR-based genome editing strategies to introduce small epitope tags into endogenous IFT172, allowing comparative analysis between antibody-based detection and direct tag visualization in living cells. For functional perturbation studies, create photo-activatable antibody fragments that can be triggered to bind IFT172 at precise spatiotemporal coordinates within cilia. Additionally, implement lattice light-sheet microscopy with adaptive optics combined with bioorthogonal chemistry approaches to visualize native IFT172 dynamics with minimal photodamage and maximum spatial resolution .