WDR92 Antibody, HRP conjugated

What is WDR92 and Why is it Important in Research?

WDR92 (WD repeat domain 92) is a highly conserved WD-repeat protein with a molecular weight of approximately 40 kDa that plays critical roles in ciliary function and protein assembly mechanisms. Research indicates that WDR92:

• Associates with prefoldin-like cochaperone complexes and known dynein assembly factors

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

• Has a phylogenetic signature consistent with roles in motile ciliary assembly or activity

• May act as a modulator of apoptosis, though this function requires further investigation

In Chlamydomonas studies, WDR92 mutants exhibit significantly impaired ciliary function, with cilia lacking both inner and outer dynein arms while maintaining intact doublet microtubules and central pairs . When WDR92 expression was knocked down in planaria, organisms showed dramatic reduction in movement capabilities due to compromised ciliary beat frequency and hydrodynamic coupling .

What are the Basic Principles of HRP Conjugation to Antibodies?

Horseradish peroxidase (HRP) is a 44 kDa glycoprotein containing approximately 18% carbohydrate content surrounding a protein core. The conjugation process typically involves:

• Generation of aldehyde groups by oxidation of carbohydrate moieties on HRP using sodium meta-periodate

• Combination of these aldehydes with amino groups on the antibody to form Schiff's bases

• Reduction using sodium cyanoborohydride to create stable covalent bonds

The resulting conjugate provides a sensitive detection system visualized through chromogenic reactions with substrates such as diaminobenzidine (DAB), ABTS, or TMB in the presence of hydrogen peroxide .

This conjugation enables detection of antigen-antibody interactions in various immunoassay applications including ELISA, western blotting, and immunohistochemistry, with conjugates functioning at dilutions ranging from 1:25 to 1:5000 depending on conjugation methodology .

How Do Different HRP-Antibody Conjugation Methods Compare in Sensitivity and Performance?

Research demonstrates significant performance differences between conjugation methodologies:

The lyophilization-enhanced method significantly improves conjugation efficiency by:

• Reducing reaction volume without changing reactant amounts

• Allowing freeze-drying of activated HRP

• Enabling longer storage of active HRP at 4°C

• Improving ability of antibodies to bind more HRP molecules

This creates essentially poly-HRP conjugates with superior sensitivity in immunoassay applications, enabling detection of lower amounts of biomarkers compared to classical methods .

What are the Critical Factors that Affect WDR92 Antibody-HRP Conjugate Stability and Performance?

Several factors critically influence the stability and performance of WDR92 antibody-HRP conjugates:

Buffer Composition Effects:

Buffer additives can significantly hamper the conjugation process, particularly when using technologies like Lightning-Link® HRP . Common buffer components that may interfere include:

• High concentrations of primary amines (Tris, glycine)

• Carrier proteins above optimal concentrations

• Sodium azide at levels above 0.1%

• Reducing agents (DTT, β-mercaptoethanol)

Storage Conditions:

Research indicates optimal storage conditions include:

• Temperature maintained at -20°C

• Addition of glycerol (typically 50%) for cryoprotection

• Inclusion of stabilizing proteins (sometimes 0.1% BSA)

• pH maintenance around 7.3

Stability Improvement:

Studies show that multi-component stabilizer systems like LifeXtend™ can protect antibody-HRP conjugates from degradation factors, preserving activity during room temperature experimentation .

How Can Researchers Optimize Western Blot Protocols Using WDR92 Antibody-HRP Conjugates?

For optimal western blot results with WDR92 antibody-HRP conjugates, consider the following protocol optimizations:

Primary Antibody Considerations:

• WDR92 antibodies typically recognize specific regions, such as amino acids 122-357 of human WDR92

• Recommended dilution ranges for western blot applications are typically 1:500-1:1000

• Confirm specificity through molecular weight verification (~40 kDa for WDR92)

Gel Filtration Analysis:

Research on WDR92 demonstrates that gel filtration chromatography using a Superose 6 10/300 column effectively separates multi-megadalton complexes, revealing associations between WDR92 and dynein components:

• In control cytoplasm, WDR92 associates with high molecular weight complexes

• In WDR92 mutants, these associations are disrupted, with altered migration patterns

This suggests careful consideration of sample preparation when studying WDR92 protein interactions.

What Experimental Approaches Best Evaluate WDR92 Function Using Antibody-HRP Conjugates?

Research indicates several effective approaches for studying WDR92 function:

Gel Filtration Fractionation:

Preparing cytoplasmic extracts through gentle freeze-thaw methods rather than mechanical disruption (vortexing with glass beads or French press) produces cleaner samples with minimal chloroplast contamination . When coupled with immunoblotting using HRP-conjugated antibodies, this approach effectively reveals:

• Protein complex formation

• Changes in oligomeric status

• Alterations in association patterns

Immunofluorescence/Immunocytochemistry:

WDR92 antibodies used at dilutions of 1:10-1:100 in immunofluorescence applications can reveal subcellular localization patterns, particularly in cells like HepG2 .

Immunohistochemistry:

For tissue sections, WDR92 antibodies have demonstrated successful detection in:

• Human colon tissue

• Human testis tissue

• Mouse testis tissue
  Recommended dilutions range from 1:50-1:1000, with antigen retrieval using TE buffer pH 9.0 or citrate buffer pH 6.0 .

How Does the Recombinant Production of HRP-Antibody Conjugates Compare with Chemical Conjugation Methods?

Recombinant production of HRP-antibody conjugates offers distinct advantages over chemical conjugation:

Key Differences:

Research demonstrates successful production of recombinant HRP-antibody conjugates using Pichia pastoris methylotrophic yeast expression systems. These conjugates maintained both enzymatic activity of HRP and antigen-binding properties of the antibody fragments .

Advanced studies show that recombinant conjugates can be designed with the antibody fragment at either the N- or C-terminus of the marker enzyme, with both configurations maintaining functional activity .

What are the Advanced Applications of WDR92 Antibody-HRP Conjugates in Ciliary Research?

WDR92 research reveals critical roles in ciliary function, making WDR92 antibody-HRP conjugates valuable tools for investigating ciliopathies and related disorders:

Dynein Preassembly Pathway Analysis:

Studies demonstrate that WDR92 functions as a specific scaffolding hub to recruit cochaperone complexes to sites of active dynein HC synthesis . Experimental approaches using WDR92 antibodies can:

• Track changes in dynein heavy chain stability

• Monitor dynein assembly factor interactions

• Characterize prefoldin-like cochaperone complex associations

Ciliopathy Research:

WDR92 knockdown studies in planaria showed pleiomorphic defects in ciliary architecture, including:

• Partial loss of dynein arms

• Incomplete closure of the B-tubule

• Occlusion or replacement of the central pair complex

These findings suggest WDR92 antibodies can help identify similar defects in other organisms or human tissue samples.

How Can Researchers Validate Specificity of WDR92 Antibody-HRP Conjugates?

Ensuring specificity of WDR92 antibody-HRP conjugates requires several validation approaches:

Molecular Verification:

• SDS-PAGE analysis to confirm expected molecular weight (40 kDa for WDR92)

• UV spectrophotometry to verify conjugation, as shown by characteristic wavelength shifts:

  • HRP typically shows a peak at 430 nm

  • Antibodies show a peak at 280 nm

  • Conjugates exhibit modified peak characteristics

Control Experiments:

• Include positive control samples with known WDR92 expression (e.g., K-562 cells)

• Include negative controls (secondary antibody only)

• Test specificity by comparing wild-type and WDR92 knockdown samples

Cross-Reactivity Testing:

Limited cross-reactivity data suggests available WDR92 antibodies may detect human and mouse WDR92, but comprehensive species testing is generally lacking in current literature .

What Future Directions Exist for WDR92 Antibody-HRP Conjugate Development and Application?

Several promising research directions emerge from current literature:

Enhanced Conjugation Methods:

The modified lyophilization approach demonstrated significantly improved sensitivity compared to classical methods, but researchers note "future exploration are necessary on wide range of IgG antibodies" , suggesting opportunities to:

• Optimize lyophilization parameters for different antibody isotypes

• Explore effects of antibody concentration on conjugation efficiency

• Investigate alternative stabilization approaches

Recombinant Technology Advancements:

Recombinant HRP-antibody conjugate production offers opportunities to:

• Design highly sensitive immunobiosensors "based on the recombinant DNA technology"

• Create targeted conjugates for specific cellular compartments or structures

• Develop standardized production methods for consistent performance

WDR92 Biological Function Exploration:

Given the demonstrated role of WDR92 in ciliary function , future research could:

• Investigate associations between WDR92 dysfunction and human ciliopathies

• Develop targeted treatments for ciliary disorders

• Explore the dual roles of WDR92 in apoptosis and ciliary function