WDR60 Antibody

What is WDR60 and what cellular functions is it involved in?

WDR60 (WD repeat domain 60) is a component of the dynein-2 complex that mediates trafficking of ciliary proteins by powering the intraflagellar transport (IFT) machinery containing IFT-A and IFT-B complexes . It functions as an intermediate chain in the dynein-2 motor complex, which is essential for proper cilia formation and function. The protein contains WD40 repeat domains in its C-terminal region (residues 627-1066) and has functional domains in its N-terminal region that facilitate interactions with various light chains of the dynein complex . WDR60 plays a critical role in retrograde transport within cilia, and disruption of its function can lead to ciliopathies.

What are the structural characteristics of WDR60 protein?

WDR60 is structurally divided into two main regions: an N-terminal region (residues 1-626) and a C-terminal WD40 domain (residues 627-1066) . The N-terminal region is responsible for interactions with dynein light chains, particularly through residues 473-522, which are crucial for binding TCTEX1D2 and DYNLT1/DYNLT3 . This region appears to be critical for protein-protein interactions within the dynein-2 complex. The C-terminal WD40 domain likely forms a β-propeller structure typical of WD-repeat proteins, which generally serve as platforms for protein complex assembly. Research has demonstrated that deletion of residues 473-522 abolishes the ability of WDR60 to interact with its light chain partners .

What types of WDR60 antibodies are available for research?

Multiple types of WDR60 antibodies are available for research applications, including polyclonal and monoclonal antibodies with various conjugations . These antibodies can be unconjugated or conjugated with tags such as HRP or APC depending on the experimental requirements . They are available in different host species, with rabbit and mouse being the most common sources . Both primary antibodies (that directly bind to WDR60) and secondary detection systems are available from commercial suppliers. Selection of the appropriate antibody should be based on specific experimental needs, including the target species, application method, and detection system.

Which applications are WDR60 antibodies most commonly used for?

WDR60 antibodies are validated for several common laboratory applications with varying degrees of optimization . Western blotting (WB) is among the most frequently validated applications, allowing researchers to detect WDR60 protein in cell or tissue lysates and analyze its expression levels or post-translational modifications . Immunohistochemistry (IHC) and immunofluorescence (IF) applications enable localization studies to determine the subcellular distribution of WDR60, particularly in relation to ciliary structures . Immunoprecipitation (IP) has been successfully employed to study protein-protein interactions involving WDR60, such as its associations with dynein light chains . ELISA applications allow for quantitative measurement of WDR60 levels in various samples .

How should WDR60 antibody be validated for ciliary protein research?

Validating WDR60 antibodies for ciliary protein research requires a multi-step approach. First, perform western blot analysis to confirm specificity using both wild-type and WDR60-knockout (WDR60-KO) cell lines as controls . For immunofluorescence validation, co-staining with established ciliary markers such as ARL13B (ciliary membrane marker) and FOP/FGFR1OP (basal body marker) is essential to confirm proper localization . The antibody should demonstrate specific staining patterns consistent with WDR60's role in the dynein-2 complex. Additionally, siRNA knockdown or CRISPR-Cas9 knockout experiments can verify specificity by showing reduced or absent signal in WDR60-depleted samples. Finally, cross-validation using different antibodies targeting distinct epitopes of WDR60 can provide increased confidence in the observed results.

What are the optimal conditions for immunoprecipitation (IP) experiments with WDR60 antibody?

For successful immunoprecipitation of WDR60 and its interaction partners, consider the following methodological approach based on published research: Use an affinity-purified WDR60 antibody at 10 μg per reaction (for antibodies similar to those offered by suppliers like Bethyl Laboratories) . Prepare cell lysates in a buffer containing 20 mM HEPES-KOH (pH 7.4), 150 mM NaCl, 0.1% Triton X-100, and protease inhibitor cocktail. Pre-clear lysates with protein A/G beads before adding the WDR60 antibody. Incubate the antibody-lysate mixture overnight at 4°C with gentle rotation, followed by addition of fresh protein A/G beads for 2-3 hours. Perform stringent washing steps (at least 4-5 washes) with lysis buffer to reduce background. For detection of interacting proteins like TCTEX1D2 or DYNLT1/DYNLT3, use the VIP (visible immunoprecipitation) assay or conventional immunoblotting with specific antibodies against these targets .

How can one investigate the interaction between WDR60 and dynein light chains?

Investigating the interaction between WDR60 and dynein light chains requires sophisticated protein-protein interaction techniques. The visible immunoprecipitation (VIP) assay has been effectively employed to demonstrate that WDR60 interacts with specific light chains, particularly TCTEX1D2, DYNLT1, and DYNLT3 . This technique involves co-expression of fluorescently tagged proteins (e.g., mCherry-WDR60 and EGFP-tagged light chains) followed by immunoprecipitation and visualization of co-precipitated proteins . For detailed mapping of interaction domains, construct deletion mutants of WDR60 (such as those lacking residues 473-522) and assess their ability to interact with light chains . Complementary approaches include yeast two-hybrid screening, proximity ligation assays, or FRET-based techniques to confirm direct interactions in different experimental contexts. Mass spectrometry analysis of immunoprecipitated complexes can further identify novel interaction partners or post-translational modifications that regulate these interactions.

What are the key considerations when comparing WDR60 and WDR34 functions in the dynein-2 complex?

When investigating the distinct yet potentially overlapping functions of WDR60 and WDR34 in the dynein-2 complex, researchers should consider several key factors. Both proteins serve as intermediate chains in the dynein-2 complex but interact with different sets of light chains: WDR60 primarily interacts with TCTEX1D2, DYNLT1, and DYNLT3, while WDR34 interacts with DYNLL2 and DYNLRB1 . Direct interaction between the WDR34 and WDR60 subcomplexes appears to be relatively weak when studied in isolation, suggesting they may function as distinct modules within the larger dynein-2 complex . When designing knockout or knockdown experiments, consider potential compensatory mechanisms between these proteins. Immunoprecipitation experiments with one intermediate chain should assess the impact on the localization and function of the other to determine if they operate independently or cooperatively. Analysis of ciliary phenotypes in single versus double knockout cell lines can provide insights into functional redundancy or unique roles of each protein in retrograde IFT processes.

How do post-translational modifications affect WDR60 function and antibody recognition?

Post-translational modifications (PTMs) of WDR60 can significantly impact both its function in the dynein-2 complex and recognition by antibodies. While the search results don't specifically address WDR60 PTMs, research on related dynein components suggests that phosphorylation, ubiquitination, and other modifications likely regulate WDR60 activity, localization, and protein interactions. When investigating PTMs, researchers should consider using phospho-specific antibodies if available, or employ phosphatase treatments of samples to assess the impact of phosphorylation on antibody recognition. Mass spectrometry analysis of immunoprecipitated WDR60 can identify specific modification sites. For functional studies, site-directed mutagenesis of potential modification sites (replacing modifiable residues with non-modifiable analogs) can help determine the impact of specific PTMs on WDR60's interaction with light chains or its localization to cilia. When selecting antibodies, review the immunogen information to determine if the epitope contains known or potential modification sites that might affect antibody binding under different cellular conditions.

What are common challenges when using WDR60 antibodies for immunofluorescence in ciliated cells?

Researchers often encounter several challenges when performing immunofluorescence with WDR60 antibodies in ciliated cells. Fixation method critically impacts epitope accessibility and antibody performance; while paraformaldehyde (PFA) is commonly used, some epitopes may require methanol fixation or a combination approach. Antibody concentration requires careful optimization; insufficient concentrations may result in weak signal, while excessive amounts can increase background. When co-staining with ciliary markers like ARL13B, carefully select compatible secondary antibodies to avoid cross-reactivity . Ciliary localization of WDR60 may be transient or dynamic, necessitating synchronization of cells or examination at various stages of ciliogenesis. Finally, antibody penetration can be challenging in structures like cilia; consider using detergents like Triton X-100 or digitonin in a graded series of concentrations to optimize permeabilization without disrupting ciliary structure.

How can specificity of WDR60 antibodies be validated in knockout systems?

Rigorous validation of WDR60 antibodies using knockout systems involves several complementary approaches. Generate WDR60-knockout cell lines using CRISPR-Cas9 or siRNA technology as negative controls . Perform side-by-side western blot analysis using wild-type and WDR60-knockout samples; a specific antibody will show a band of the expected molecular weight (approximately 122 kDa) in wild-type samples that is absent in knockout samples . For immunofluorescence validation, conduct parallel staining of wild-type and knockout cells, expecting loss of specific signal in knockout cells while maintaining other cellular staining patterns . Rescue experiments, where WDR60 expression is restored in knockout cells, provide additional validation by demonstrating reappearance of antibody signal. Finally, cross-validation with multiple antibodies targeting different regions of WDR60 can confirm specificity - all should show concordant loss of signal in knockout systems despite recognizing different epitopes.

What factors influence WDR60 antibody selection for studying its interaction with TCTEX1D2?

When selecting a WDR60 antibody to study its interaction with TCTEX1D2, researchers must consider several critical factors. The epitope recognized by the antibody is paramount; antibodies targeting the N-terminal region (specifically residues 473-522) may interfere with or be blocked by the interaction with TCTEX1D2, as this region is crucial for binding light chains . Conversely, antibodies targeting the C-terminal WD40 domain would be less likely to disrupt these interactions. The antibody's suitability for immunoprecipitation is essential, as this technique is frequently used to pull down protein complexes; verification of IP capability should be confirmed before proceeding with interaction studies . Additionally, consider using antibodies that have been explicitly validated for detecting native protein complexes rather than denatured proteins. For co-immunoprecipitation experiments, combinations of antibodies raised in different host species (e.g., rabbit anti-WDR60 and mouse anti-TCTEX1D2) will facilitate simultaneous detection without cross-reactivity issues.

How are WDR60 antibodies being used to investigate ciliopathies?

WDR60 antibodies are valuable tools for investigating ciliopathies, which are genetic disorders resulting from ciliary dysfunction. Studies utilize these antibodies to examine WDR60 localization and expression in patient-derived cells, particularly fibroblasts or induced pluripotent stem cells differentiated into ciliated cell types . Immunofluorescence microscopy with WDR60 antibodies, combined with markers like ARL13B (ciliary membrane) and FOP/FGFR1OP (basal body), can reveal abnormal ciliary phenotypes in patient samples . Western blotting with WDR60 antibodies enables quantification of protein expression levels in affected tissues, potentially identifying cases where mutations lead to protein instability rather than functional defects. Immunoprecipitation experiments using WDR60 antibodies can detect aberrant interactions between WDR60 and its binding partners in ciliopathy models, providing mechanistic insights into disease pathogenesis. Additionally, these antibodies facilitate screening of potential therapeutic compounds by monitoring their effects on WDR60 localization and function in disease models.

What methodological approaches combine WDR60 antibodies with live cell imaging techniques?

Advanced research combining WDR60 antibodies with live cell imaging requires specialized methodological approaches. While conventional antibodies cannot penetrate live cells, researchers can transfect cells with fluorescently tagged nanobodies derived from WDR60 antibodies, enabling real-time visualization of WDR60 dynamics. Alternatively, express fluorescently tagged WDR60 (such as mCherry-WDR60) in cells to track its movement and validate observations with fixed-cell antibody staining . For ciliary transport studies, perform Fluorescence Recovery After Photobleaching (FRAP) experiments with tagged WDR60, followed by antibody staining to correlate dynamic behavior with interaction partner localization. Single-molecule tracking approaches can be used with quantum dot-conjugated antibody fragments in semi-permeabilized cells to analyze WDR60 movement along microtubules. Finally, correlative light and electron microscopy (CLEM) combines live cell imaging of fluorescently tagged WDR60 with subsequent immunogold labeling using WDR60 antibodies for ultrastructural localization, providing multi-scale analysis of WDR60 function.

How can WDR60 antibodies be employed in high-throughput screening for dynein-2 complex modulators?

WDR60 antibodies can be strategically deployed in high-throughput screening campaigns to identify compounds that modulate the dynein-2 complex function. Develop automated immunofluorescence assays that measure WDR60 localization to cilia using high-content imaging systems, with proper ciliary localization as the readout for functional dynein-2 complex . Create ELISA-based protein interaction assays using immobilized WDR60 antibodies to capture the protein and detect binding to known partners like TCTEX1D2, DYNLT1, or DYNLT3; compounds that enhance or disrupt these interactions would be potential modulators . Establish cellular thermal shift assays (CETSA) with WDR60 antibodies to identify compounds that directly bind to and stabilize WDR60 structure. For functional screens, develop reporter cell lines where ciliary trafficking defects (caused by dynein-2 dysfunction) activate expression of fluorescent or luminescent proteins, then validate hits by examining their effects on WDR60-light chain interactions using co-immunoprecipitation with validated antibodies . Finally, implement multiplexed screening where WDR60 antibodies are used alongside antibodies against other dynein-2 components to simultaneously assess effects on multiple aspects of complex assembly and function.