IFT88 Antibody

What is IFT88 and why is it important in research?

IFT88 is an essential component of the intraflagellar transport complex B (IFT-B), which mediates anterograde transport along cilia and flagella. This protein plays a crucial role in ciliogenesis and ciliary function, making it a valuable research target for studying ciliopathies, developmental disorders, and cellular signaling pathways . IFT88 is highly conserved across species, with homologs identified in organisms ranging from Chlamydomonas to humans, indicating its fundamental importance in eukaryotic cellular function . Studies have demonstrated that loss of IFT88 results in cilia dysfunction and can lead to significant developmental abnormalities and pathological conditions in various organ systems.

Which applications can IFT88 antibodies be used for?

IFT88 antibodies can be utilized across multiple experimental applications. Western blotting (WB) can be performed at dilutions ranging from 1:2000 to 1:12000, depending on the specific antibody and sample . Immunoprecipitation (IP) typically requires 0.5-4.0 μg of antibody for 1.0-3.0 mg of total protein lysate . Immunohistochemistry (IHC) works effectively at dilutions between 1:200 and 1:800 . Immunofluorescence (IF) and immunocytochemistry (ICC) applications generally use similar dilution ranges of 1:200-1:800 . Additional applications include ELISA, flow cytometry, and co-immunoprecipitation, though optimal conditions should be determined empirically for each experimental system .

What species reactivity do IFT88 antibodies demonstrate?

Commercial IFT88 antibodies exhibit cross-reactivity across multiple species. The Proteintech antibody (13967-1-AP) has confirmed reactivity with human, mouse, rat, and canine samples . Published literature has cited successful application with additional species including pig, chicken, and zebrafish . The antibody from antibodies-online (ABIN185555) demonstrates cross-reactivity with dog, human, mouse, and rat samples . When selecting an antibody for your experiment, it's important to verify the validated species reactivity for your specific model organism to ensure reliable results.

What is the molecular weight of IFT88 in Western blot applications?

IFT88 has a calculated molecular weight of approximately 94 kDa, which aligns with the observed molecular weight in Western blot applications . The anti-IFT88 antibody typically recognizes a single protein band of approximately 90 kDa, consistent with the predicted mass of 92.9 kDa for IFT88 . This consistency between calculated and observed molecular weights provides confidence in antibody specificity when conducting Western blot experiments.

How should I optimize IFT88 antibody dilutions for different experimental applications?

Optimization of antibody dilutions is critical for obtaining specific signals while minimizing background. For Western blot applications, begin with a mid-range dilution (1:5000) and adjust based on signal intensity and background levels . For immunohistochemistry and immunofluorescence, start with 1:400 dilution and modify as needed . It's advisable to perform a dilution series (e.g., 1:200, 1:400, 1:800) to determine optimal conditions for your specific tissue or cell type. For challenging applications, consider signal amplification methods or increased antibody concentration along with extended incubation times. Always include appropriate positive and negative controls to validate specificity, such as IFT88 knockout cells compared with wild-type cells .

What antigen retrieval methods are recommended for IFT88 immunohistochemistry?

Effective antigen retrieval is essential for IFT88 detection in fixed tissues. The recommended primary method is heat-induced epitope retrieval using TE buffer at pH 9.0 . Alternatively, citrate buffer at pH 6.0 can be used when TE buffer yields suboptimal results . For particularly challenging samples such as mature sperm cells, more aggressive antigen retrieval protocols may be necessary, similar to those used for enhancing both IFT88 and tubulin labeling in photoreceptor outer segments . The specific fixation method employed can significantly impact antibody accessibility to the epitope, so optimization of both fixation and antigen retrieval protocols may be required for consistent results.

How can I validate the specificity of IFT88 antibodies in my experimental system?

Validating antibody specificity is crucial for generating reliable data. Include positive controls such as tissues or cell lines known to express IFT88 (e.g., HEK-293 cells, NIH/3T3 cells, MDCK cells, or mouse thymus tissue) . Incorporate negative controls by using IFT88 knockout samples or siRNA knockdown cells, which should show significantly reduced or absent signal . When possible, use multiple antibodies targeting different epitopes of IFT88 to confirm consistent staining patterns. Western blot verification should demonstrate a single band at the expected molecular weight of approximately 94 kDa . For immunofluorescence studies, co-localization with other ciliary markers can provide additional validation of specificity.

How can IFT88 antibodies be used to study ciliary transport mechanisms?

Investigating ciliary transport mechanisms requires sophisticated approaches combining live-cell imaging with fixed-cell immunofluorescence. For quantitative analysis of IFT88 transport, establish stable cell lines expressing fluorescently tagged IFT88 and use live-cell imaging to track particle movement along cilia . Fixed-cell immunofluorescence with IFT88 antibodies at 1:200-1:800 dilutions can reveal the distribution pattern of IFT88 within cilia . Recent studies have measured anterograde IFT88 train lengths, finding an average of 358 nm (ranging from 152–650 nm), corresponding to approximately 58 IFT-B particles per train . This approach can be combined with super-resolution microscopy techniques to overcome the diffraction limit and achieve more precise measurements of IFT train composition and dynamics.

What are the considerations for using IFT88 antibodies in developmental studies?

Developmental studies require careful attention to temporal expression patterns of IFT88. During spermatogenesis, IFT88 expression shows dynamic regulation, with strong labeling in the heads and developing flagella of step 2-3 through 11 spermatids . By step 15, IFT88 labeling becomes less intense and concentrates in the cytoplasmic lobe and proximal principal piece, suggesting downregulation as the tail reaches maturity . IFT88 is not detected in mature sperm from the caudal epididymides, even with enhanced antigen retrieval methods . For developmental studies, it's crucial to select appropriate fixation methods that preserve both tissue architecture and epitope accessibility. Collection of samples at defined developmental timepoints will ensure capture of transient expression patterns that might be missed with endpoint analysis.

How can IFT88 antibodies be used to investigate the relationship between cilia and disease pathogenesis?

Investigating cilia in disease contexts requires careful experimental design. For endothelial-to-mesenchymal transition (EndMT) studies, combine IFT88 immunostaining with endothelial markers (CD31, Tie-2, VE-cadherin) and mesenchymal markers (αSMA, N-Cadherin, FSP-1) . When studying fibrosis models, consider both in vitro approaches using siRNA-mediated IFT88 silencing in endothelial cells and in vivo models using tissue-specific knockout mice (e.g., Ift88 endo mice) . In bleomycin-induced pulmonary fibrosis models, loss of endothelial Ift88 exacerbates fibrotic responses, highlighting the importance of cilia in maintaining endothelial cell identity and preventing pathological EndMT . Dual immunofluorescence staining with IFT88 and pathway-specific markers (such as Sonic Hedgehog signaling components) can reveal mechanistic insights into how ciliary dysfunction contributes to disease progression.

What are common troubleshooting strategies for weak or non-specific IFT88 antibody signals?

When encountering weak signals, several strategies can improve detection. Increase antibody concentration gradually while monitoring background levels, or extend primary antibody incubation time to overnight at 4°C . For formalin-fixed tissues, optimize antigen retrieval methods by comparing TE buffer (pH 9.0) with citrate buffer (pH 6.0) . If background is problematic, implement additional blocking steps using 5% BSA or normal serum from the same species as the secondary antibody. For tissues with high autofluorescence, consider using Sudan Black B treatment or specialized quenching reagents. In Western blot applications, transfer efficiency can significantly impact signal strength, so optimize transfer conditions for high molecular weight proteins and consider using gradient gels for better resolution around the 94 kDa size range of IFT88 .

How should samples be prepared to preserve IFT88 epitopes for antibody detection?

Sample preparation is critical for maintaining epitope integrity. For cell culture samples, 4% paraformaldehyde fixation for 10-15 minutes at room temperature preserves most epitopes while maintaining cell morphology . For tissue samples, shorter fixation times (24 hours or less) in 4% paraformaldehyde are preferable to extended fixation, which can mask epitopes . Fresh frozen tissues often provide superior antigenicity compared to formalin-fixed paraffin-embedded samples. When using paraffin embedding, ensure complete dehydration and paraffin infiltration to prevent sectioning artifacts. For immunofluorescence of ciliated structures, gentle permeabilization with 0.1-0.2% Triton X-100 is recommended to maintain ciliary architecture while allowing antibody access . Storage of fixed samples should be at -20°C in a suitable buffer system with 50% glycerol to prevent epitope degradation over time .

How can I effectively use IFT88 antibodies in co-immunoprecipitation experiments?

Co-immunoprecipitation (Co-IP) experiments require careful optimization to preserve protein-protein interactions. Use gentle lysis buffers containing 0.5-1% NP-40 or Triton X-100 with protease inhibitors to maintain complex integrity . For IFT88 Co-IP, 0.5-4.0 μg of antibody is typically required for 1.0-3.0 mg of total protein lysate . Pre-clear lysates with appropriate control IgG and protein A/G beads to reduce non-specific binding. Incubate antibody with lysate overnight at 4°C with gentle rotation to maximize binding while minimizing degradation. Include appropriate negative controls such as IgG from the same species as the IFT88 antibody. When eluting complexes, avoid harsh conditions that might disrupt interactions; instead, use non-denaturing elution buffers or competitive elution with immunizing peptide. Western blot analysis of both input and IP fractions should be performed to assess enrichment efficiency and specificity.

How can IFT88 antibodies be used in studies of ciliary signaling pathways?

Investigating ciliary signaling requires sophisticated experimental approaches. Combine IFT88 immunofluorescence with co-staining for signaling components such as Sonic Hedgehog pathway effectors, which show increased expression following loss of IFT88 in endothelial cells . For dynamic pathway studies, use synchronized cell populations to capture temporal changes in ciliary localization of signaling proteins. Recent research has revealed that IFT88 contributes to the ciliary localization of TRPV channels, suggesting a broader role in sensory signaling beyond structural transport . Quantitative image analysis should include measurements of co-localization coefficients and signal intensity profiles along the ciliary axis to identify compartmentalization of signaling components.

What are the considerations for using IFT88 antibodies in tissue-specific knockout models?

When working with tissue-specific knockout models like Ift88 endo mice, verification of knockout efficiency is essential. Isolate the relevant cell population (e.g., cardiac and pulmonary endothelial cells) and confirm loss of Ift88 expression using both protein detection (Western blot, immunofluorescence) and transcript analysis (qPCR) . Include rigorous controls such as Cre-negative littermates and cells from non-targeted tissues to confirm specificity of knockout. When phenotyping, consider both baseline conditions and stress challenges (such as bleomycin treatment or aortic banding) to reveal conditional phenotypes that may not be apparent under normal conditions . For comprehensive analysis, combine protein-level studies using IFT88 antibodies with functional assays relevant to the tissue of interest, such as barrier function tests for endothelial cells or contractility measurements for cardiac tissue.

How can advanced imaging techniques enhance IFT88 antibody applications in cilia research?

Advanced imaging approaches can significantly extend the utility of IFT88 antibodies. Super-resolution microscopy techniques (STED, STORM, SIM) can overcome the diffraction limit to resolve IFT88 localization within the 250 nm diameter of the cilium . For studies of IFT dynamics, combine immunofluorescence of fixed samples with live imaging of fluorescently tagged IFT88 to correlate static localization patterns with dynamic transport processes. Expansion microscopy can physically enlarge specimens to reveal nanoscale organization of IFT complexes beyond the resolution limit of conventional microscopy. Light sheet microscopy offers advantages for imaging IFT88 in thick tissue samples or whole model organisms with minimal photobleaching. For quantitative analysis, implement automated image processing workflows to measure parameters such as ciliary length, IFT88 signal intensity along the cilium, and co-localization with other ciliary proteins across large datasets.