WDR92 Antibody, Biotin conjugated

What is WDR92 and what are its known biological functions?

WDR92 (WD repeat domain 92) is a highly conserved WD-repeat protein with multiple cellular functions. Research has identified several critical roles:

• Acts as a modulator of apoptosis through cellular signaling pathways

• Functions as a component of a prefoldin-like cochaperone complex involved in protein folding

• Plays a crucial role in motile cilia assembly and activity

• Serves as a key assembly factor specifically required for the stability of axonemal dynein heavy chains in cytoplasm

Studies in organisms like Chlamydomonas and planaria have demonstrated that WDR92 is essential for proper ciliary function. When WDR92 expression is knocked down in planaria, researchers observed ciliary loss, reduced beat frequency, and dyskinetic motion of remaining ventral cilia . In Chlamydomonas, the wdr92-1 mutant (missing the last four WD repeats) builds only ~0.7-μm cilia lacking both inner and outer dynein arms, while maintaining intact doublet microtubules and central pair structures .

What are the characteristics of biotin-conjugated WDR92 antibodies?

Biotin-conjugated WDR92 antibodies are specialized immunological tools with the following characteristics:

• Polyclonal antibodies typically raised in rabbits against recombinant WDR92 protein (amino acids 122-357 of human WDR92)

• Conjugated with biotin molecules via chemical linkage for detection purposes

• Preserved in solution containing 0.03% Proclin 300, 50% Glycerol, 0.01M PBS at pH 7.4

• Designed primarily for ELISA applications in research settings

• Recognize aliases including FLJ31741, Monad, WD repeat-containing protein 92, and WD repeat-containing protein Monad

• Demonstrate reactivity against human WDR92 protein

The biotin conjugation provides significant advantages in experimental applications due to the strong, specific interaction between biotin and streptavidin, which forms quickly and remains stable under varying pH and temperature conditions .

How should biotin-conjugated WDR92 antibodies be implemented in ELISA protocols?

When using biotin-conjugated WDR92 antibodies in ELISA, researchers should follow this methodological approach:

• Plate preparation: Coat wells with capture antibody (for sandwich ELISA) or WDR92 antigen (for direct ELISA) at 1-10 μg/ml in carbonate buffer

• Blocking: Block non-specific binding sites with 1-5% BSA or non-fat milk for 1-2 hours at room temperature

• Sample addition: Add diluted samples containing potential WDR92 protein

• Detection: Apply biotin-conjugated WDR92 antibody at manufacturer-recommended dilution (typically 1:1000-1:5000)

• Signal development: Add streptavidin-HRP conjugate followed by appropriate substrate

• Analysis: Measure optical density and calculate results against standards

It's important to note that competitive immunoassay formats are particularly susceptible to biotin interference, with elevated biotin levels potentially causing significant signal distortion . Even a 10 ng/ml increase in biotin concentration can result in a 20.8% average increase in measured values, rising to 50.1% average increase at 150 ng/ml biotin elevation .

What methods can be used to study WDR92's role in ciliary assembly and function?

Based on published research, several methodological approaches have proven effective for investigating WDR92's role in ciliary assembly:

• Genetic manipulation techniques:

  • RNAi-based knockdown (as employed in planaria studies)

  • Characterization of insertional mutations (as in Chlamydomonas)

  • Generation of double mutants (e.g., wdr92-1 tpg1-2 in Chlamydomonas)

• Biochemical analysis methods:

  • Cytoplasmic extract preparation via gentle freeze/thaw cycles

  • Gel filtration chromatography for protein complex separation

  • Immunoblotting to detect dynein components and their distribution

  • Mass spectrometry for comprehensive protein complex analysis

• Functional and structural assessments:

  • Transmission electron microscopy to examine ciliary ultrastructure

  • High-speed videomicroscopy to quantify ciliary beat frequency

  • Motility assays to assess organism movement capabilities

Table 1: Comparison of WDR92 Mutation/Knockdown Effects Across Model Organisms

How can biotin interference be prevented in immunoassays using biotin-conjugated antibodies?

Biotin interference represents a significant technical challenge when using biotin-conjugated antibodies. Researchers should implement these strategies to minimize interference:

• Sample preparation protocols:

  • Pre-treat samples with streptavidin-coated microparticles to sequester free biotin

  • Dilute samples when possible to reduce biotin concentration

  • Use commercial biotin blocking systems that employ specific blocking reagents

• Assay design considerations:

  • Implement sandwich ELISA formats which demonstrate lower susceptibility to biotin interference than competitive formats

  • Include interference-suppressed immunoassay components when available

  • Apply mathematical correction factors based on known biotin concentrations

• Validation approaches:

  • Run parallel assays with non-biotin detection systems for result confirmation

  • Include control samples with known biotin concentrations (10, 30, 50, 100, and 150 ng/ml)

  • Measure biotin levels in test samples using dedicated biotin quantification assays

Research has demonstrated that competitive immunoassays show progressive signal distortion with increasing biotin concentrations, with values increasing by 35.8% on average at biotin elevations of 100 ng/ml .

What storage and handling practices optimize biotin-conjugated WDR92 antibody performance?

To maintain optimal functionality of biotin-conjugated WDR92 antibodies, researchers should adhere to these evidence-based practices:

• Storage conditions:

  • Store antibody at -20°C or -80°C upon receipt

  • Avoid repeated freeze-thaw cycles by preparing single-use aliquots

  • Maintain in buffer containing stabilizers (50% glycerol) and preservatives (0.03% Proclin 300)

• Working practices:

  • Thaw antibody aliquots on ice immediately before use

  • Centrifuge briefly before opening to collect solution at the bottom of the tube

  • Return to -20°C promptly after use

• Dilution recommendations:

  • Prepare working dilutions fresh on the day of experiment

  • Use high-quality, low-protein-binding tubes for dilution

  • Dilute in buffers matching the experimental application (PBS with 1% BSA for ELISA)

These practices help preserve the structural integrity of both the antibody and the biotin conjugate, ensuring consistent experimental performance and reproducible results.

How does WDR92 contribute to axonemal dynein assembly pathways?

WDR92 plays a crucial and specific role in the assembly pathway for axonemal dyneins, particularly affecting heavy chains. Biochemical and genetic analyses reveal:

• Molecular mechanism insights:

  • WDR92 associates with prefoldin-like cochaperone complexes and established dynein assembly factors

  • It interacts with RPAP3 (RNA polymerase II-associated protein 3), likely through the RPAP3_C domain

  • This interaction domain is also found in two known ciliary dynein assembly factors: CCDC103 and SPAG1

• Evidence from mutant analyses:

  • Chlamydomonas wdr92-1 cytoplasmic extracts show near-complete absence of all three outer arm dynein heavy chains

  • The IFT dynein heavy chain remains present in normal amounts, suggesting pathway specificity

  • Outer arm dynein intermediate chains (ICs) and light chains (LCs) shift to low molecular weight fractions but remain present

  • The oligomeric status of DYX1C1/PF23 (DNAAF4) assembly factor is significantly altered in wdr92-1 mutants

Current models suggest WDR92 functions within a specialized cytoplasmic chaperone system specifically required for folding key components of motile ciliary axonemes, with particular importance for the stable synthesis of dynein heavy chains .

What genetic and molecular evidence supports WDR92's evolutionary conservation and specialized function?

WDR92 demonstrates remarkable evolutionary conservation with a phylogenetic distribution pattern strongly correlated with motile cilia presence:

• Structural conservation analysis:

  • WDR92 contains highly conserved WD-repeat domains forming β-propeller structures

  • These domains serve as protein-protein interaction platforms critical for chaperone function

  • Even partial disruption (loss of last four WD repeats in Chlamydomonas) causes severe ciliary defects

• Functional conservation evidence:

  • WDR92 knockdown in planaria results in pleiomorphic defects in ciliary architecture

  • These include partial loss of dynein arms, incomplete B-tubule closure, and central pair complex abnormalities

  • Similar ciliary assembly defects occur in Chlamydomonas wdr92 mutants, though with organism-specific variations

• Protein interaction network conservation:

  • WDR92 interacts with components of the R2TP complex (RuvBL1/2, PIHD1, RPAP3)

  • It also associates with prefoldin cochaperone complex members across diverse species

  • These interactions place WDR92 within a broadly conserved protein quality control system

The high degree of conservation across evolutionarily distant organisms possessing motile cilia, combined with its absence in organisms lacking these structures, strongly supports WDR92's specialized role in ciliary assembly across eukaryotes.

How can researchers distinguish between WDR92's direct and indirect effects in experimental systems?

Distinguishing direct from indirect effects of WDR92 requires sophisticated experimental approaches:

• Biochemical interaction analyses:

  • Perform co-immunoprecipitation with biotin-conjugated WDR92 antibodies followed by mass spectrometry

  • Conduct yeast two-hybrid or proximity labeling experiments to identify direct binding partners

  • Use recombinant protein interaction assays with purified components to confirm direct interactions

• Genetic rescue experiments:

  • Express truncated or domain-mutated WDR92 variants in wdr92 mutant backgrounds

  • Implement structure-function analysis using chimeric proteins with domains from related WD-repeat proteins

  • Create targeted mutations in specific interaction domains to disrupt select protein partnerships

• Temporal analysis approaches:

  • Apply inducible knockdown/knockout systems to study acute versus chronic loss of WDR92

  • Use pulse-chase experiments to track protein synthesis and stability in the presence/absence of WDR92

  • Implement live imaging of fluorescently tagged dynein components during ciliary assembly

Research indicates that WDR92 specifically affects axonemal dynein heavy chain stability without directly impacting intermediate chains, light chains, or IFT dynein . This specificity provides a valuable experimental framework for distinguishing direct effects on dynein assembly from indirect consequences for ciliary structure and function.

What are the emerging research directions for WDR92 beyond ciliary assembly?

While WDR92's role in ciliary assembly is well-established, several emerging research areas warrant investigation:

• Potential roles in apoptotic pathways:

  • Early research identified WDR92/Monad as a proapoptotic factor based on overexpression studies

  • The relationship between this function and ciliary assembly roles remains unexplored

  • Investigation of cell-type specific expression patterns may reveal context-dependent functions

• Connections to RNA polymerase assembly:

  • WDR92 interacts with several components of prefoldin complexes that aid RNA polymerase II assembly

  • This suggests potential roles in transcriptional regulation beyond ciliary functions

  • Comparative studies across cell types with and without cilia could clarify these functions

• Implications for human ciliopathies:

  • WDR92 mutations may contribute to human disorders involving motile cilia dysfunction

  • Screening ciliopathy patient cohorts for WDR92 variants could identify previously unrecognized genetic causes

  • Development of model systems harboring patient-specific mutations would advance understanding of pathogenic mechanisms

These research directions highlight the need for continued investigation of WDR92's multifaceted cellular functions beyond its established role in ciliary dynein assembly.