WDR73 Antibody, HRP conjugated

What is WDR73 and why is it relevant to study?

WDR73 is a WD40-repeat-containing protein that plays crucial roles in multiple cellular processes. It contains six WD40 motifs which function as scaffolds for protein complex assembly . WDR73 is particularly significant because:

• It is expressed in the brain and kidney

• It undergoes dynamic localization during the cell cycle, appearing diffuse in the cytoplasm during interphase but relocating to spindle poles and astral microtubules during mitosis

• Loss-of-function mutations in WDR73 cause Galloway-Mowat syndrome (GAMOS), a rare neurodegenerative disorder characterized by neurological defects and renal-glomerular disease

• It interacts with the INTS9 and INTS11 components of the Integrator complex, implicating it in RNA metabolism and transcriptional control pathways

Understanding WDR73's function has significant implications for both basic cell biology and clinical research related to developmental disorders.

For optimal detection of WDR73 using HRP-conjugated antibodies, sample preparation is critical:

For cell/tissue preparations:

• Fix cells using either cold 100% methanol or 4% paraformaldehyde (PFA) depending on the epitope accessibility

• For PFA-fixed cells, perform antigen retrieval with NH₄Cl treatment

• For tissue sections, perform antigen retrieval with TE buffer (pH 9.0) or citrate buffer (pH 6.0)

• Block with appropriate normal serum (typically 10% in PBS) for 30 minutes to prevent non-specific binding

• Primary antibody incubation should be performed overnight at 4°C for optimal binding

Buffer considerations:

• The antibody is typically stored in PBS with preservatives (such as 0.03% Proclin 300) and stabilizers (such as 50% glycerol)

• Working dilutions should be prepared in a buffer matching your application (typically PBS with 1-5% BSA)

How can I validate the specificity of WDR73 Antibody, HRP conjugated in my experimental system?

Validating antibody specificity is crucial for accurate results. For WDR73 Antibody, HRP conjugated:

• Positive controls: Use tissues known to express WDR73, such as:

  • Human, mouse, or rat kidney tissue

  • Human, mouse, or rat brain/cerebellum tissue

• Negative controls:

  • Omit primary antibody while maintaining all other steps

  • Use tissues from WDR73 knockout models (CRISPR/Cas9 system targeting WDR73 has been successfully used)

• Molecular weight verification:

  • The observed molecular weight of WDR73 is approximately 41 kDa

  • In Western blot applications, verify band size against this standard

• Peptide competition assay:

  • Pre-incubate the antibody with the immunizing peptide (the antibody targets AA 163-366 of WDR73)

  • If signal disappears, this confirms specificity

• Cross-validation:

  • Compare results with alternative WDR73 antibodies targeting different epitopes

What are the technical challenges in detecting WDR73 in different subcellular compartments?

Detecting WDR73 presents unique challenges due to its dynamic subcellular localization:

How can WDR73 Antibody, HRP conjugated be used to investigate Galloway-Mowat syndrome pathogenesis?

WDR73 mutations are causative of Galloway-Mowat syndrome (GAMOS), a rare disorder with neurological defects and renal disease. Using WDR73 Antibody, HRP conjugated in GAMOS research:

• Patient sample analysis:

  • Compare WDR73 protein levels in patient tissues versus controls

  • Analyze expression patterns in affected tissues (brain, kidney)

  • Determine if truncated proteins are produced from mutant alleles

• Functional studies in cell models:

  • Create WDR73-deficient cell lines using CRISPR/Cas9 (targeting strategies have been published)

  • Assess microtubule network alterations, as fibroblasts from affected children display abnormal nuclear morphology and microtubule network disturbances

  • Monitor cell viability, as WDR73 depletion leads to reduced cell survival

• Mechanistic investigations:

  • Study WDR73's role in Integrator complex function and UsnRNA processing

  • Examine transcriptional responses to growth factor stimulation, which are altered in WDR73-deficient cells

  • Investigate cell cycle progression defects, as WDR73-deficient cells show G2/M phase arrest

• Podocyte-specific studies:

  • Examine focal adhesion formation, as WDR73 depletion destabilizes PIP4K2C activity and impairs focal adhesion

  • Analyze podocyte morphology and function in WDR73-deficient models

What methodological approaches can overcome potential false negatives when detecting mutant WDR73 proteins?

Detecting mutant WDR73 proteins presents challenges, especially with truncated proteins:

• Epitope considerations:

  • The commercially available HRP-conjugated WDR73 antibody targets amino acids 163-366

  • This may miss truncated proteins from mutations like p.Tyr43* (causing premature termination)

  • For comprehensive analysis, use antibodies targeting different regions of WDR73

• Multiple detection methods:

  • Combine protein detection (antibody-based) with mRNA analysis (RT-PCR, RNA-seq)

  • Use mass spectrometry for unbiased protein identification

• Protein stabilization strategies:

  • Treat cells with proteasome inhibitors (MG132) to prevent rapid degradation of unstable mutant proteins

  • Explore autophagy inhibitors, as WDR73 regulates PIP4K2C stability through the autophagy-lysosomal pathway

• Alternative constructs:

  • Express tagged versions of mutant WDR73 (N-terminal HA tags have been successfully used)

  • Monitor tagged protein localization and stability

How can WDR73 antibodies be employed in high-throughput screens for therapeutic compounds targeting Galloway-Mowat syndrome?

For drug discovery targeting GAMOS, WDR73 antibodies can enable several screening approaches:

• Phenotypic rescue assays:

  • Establish high-content imaging system using WDR73-deficient cells

  • Monitor rescue of phenotypes (nuclear morphology, microtubule organization, focal adhesion)

  • Use HRP-conjugated antibodies in automated ELISA-based detection systems

• Pathway-specific screens:

  • Target the Integrator complex pathway and UsnRNA processing

  • Screen for compounds affecting transcriptional responses to growth factors

  • Focus on cell cycle regulatory proteins, as WDR73 suppression alters expression of these genes

• PIP4K2C stabilization assays:

  • Develop assays monitoring PIP4K2C protein levels using WDR73 and PIP4K2C antibodies

  • Screen for compounds that stabilize PIP4K2C in WDR73-deficient cells

  • Measure restoration of focal adhesion formation

• Technical considerations:

  • Optimize signal-to-noise ratio for HRP detection in high-throughput formats

  • Develop robust quality control metrics using positive controls (known WDR73 interactors)

  • Consider multiplexed assays that simultaneously measure multiple endpoints

What are the common causes of inconsistent results when using WDR73 Antibody, HRP conjugated?

When experiencing variable results with WDR73 Antibody, HRP conjugated, consider these common issues:

• Cell cycle variability:

  • WDR73 localization changes throughout the cell cycle

  • Unsynchronized cell populations may show variable staining patterns

  • Solution: Synchronize cells or use cell cycle markers to categorize results

• Epitope accessibility issues:

  • WDR73's interaction with other proteins may mask epitopes

  • Solution: Try multiple fixation and permeabilization protocols

• Technical factors:

  • Storage conditions affect antibody stability (store at -20°C with 50% glycerol)

  • HRP activity is sensitive to azide and metal ions

  • Solution: Prepare fresh dilutions for each experiment

• Sample-specific considerations:

  • Expression levels vary between tissues (higher in brain and kidney)

  • Solution: Use appropriate positive controls for standardization

How can I optimize WDR73 detection in complex kidney tissue samples for studying podocyte pathology?

For optimal detection of WDR73 in kidney tissues, especially for podocyte studies:

• Tissue processing optimization:

  • Fresh tissue fixation in 4% PFA followed by paraffin embedding preserves structure

  • For frozen sections, snap-freeze in OCT compound

  • Section thickness of 4-6 μm is optimal for antibody penetration

• Antigen retrieval methods:

  • Heat-induced epitope retrieval using TE buffer (pH 9.0) is recommended

  • Alternative: citrate buffer (pH 6.0) may work for some epitopes

  • Optimize heating time and temperature (typically 95-100°C for 20-30 minutes)

• Specific podocyte staining:

  • Double labeling with podocyte markers (nephrin, podocin) helps identify specific cells

  • For confocal microscopy, use separate fluorophore-conjugated secondary antibodies instead of HRP

  • For brightfield microscopy, sequential staining with careful color development

• Background reduction strategies:

  • Block endogenous peroxidase activity with H₂O₂ treatment (3% for 30 min)

  • Use avidin-biotin blocking if using biotin-based detection systems

  • Increase blocking serum concentration to 10% if background persists

How do different fixation methods affect WDR73 epitope preservation and detection?

Fixation significantly impacts WDR73 detection, with different methods preserving distinct aspects of its biology:

For WDR73 Antibody, HRP conjugated, testing multiple fixation methods during protocol optimization is advised to determine which best preserves the specific epitope (AA 163-366) in your experimental system.

How can WDR73 antibodies contribute to understanding shared pathogenic mechanisms between neurodevelopmental disorders and kidney diseases?

WDR73 research offers unique insights into mechanisms shared between neural and renal tissues:

• Cellular similarities exploration:

  • Both neurons and podocytes are post-mitotic cells with elaborate cytoskeletons

  • Use WDR73 antibodies to compare cytoskeletal organization and microtubule dynamics

  • Examine focal adhesion structures, which are critical for both neuronal migration and podocyte function

• Developmental timing studies:

  • Track WDR73 expression during embryonic development of both systems

  • Compare with timing of pathological changes in Galloway-Mowat syndrome

  • Use tissue-specific conditional knockout models to determine critical developmental windows

• Integrator complex function analysis:

  • Study WDR73's interaction with INTS9 and INTS11 in both neural and renal tissues

  • Compare UsnRNA processing efficiency between tissues

  • Examine tissue-specific transcriptional responses to signaling factors

• Methodological approach:

  • Use identical antibody-based protocols across tissues for direct comparisons

  • Perform systematic co-immunoprecipitation studies to identify tissue-specific interaction partners

  • Develop organoid models of both neural and renal tissues with WDR73 mutations

What experimental approaches can reconcile contradictory findings about WDR73 function in different cellular contexts?

To address discrepancies in WDR73 research across different cellular systems:

• Standardized cellular models:

  • Establish a panel of cell lines with consistent WDR73 manipulation (knockout, knockdown, overexpression)

  • Include both dividing cells and differentiated post-mitotic cells

  • Use inducible systems to control timing of WDR73 depletion

• Comprehensive functional assessment:

  • Systematically evaluate all reported WDR73 functions:

    • Microtubule organization and dynamics

    • Cell cycle progression

    • UsnRNA processing

    • Transcriptional regulation

    • PIP4K2C stability and focal adhesion

  • Document context-specific differences

• Protein interaction mapping:

  • Use proximity labeling approaches (BioID, TurboID) to map WDR73 interaction networks

  • Compare interaction partners between different cell types

  • Verify key interactions with co-immunoprecipitation using WDR73 antibodies

• Resolution strategies for contradictory findings:

  • Determine if contradictions are due to cell-type specificity, WDR73 isoforms, or experimental conditions

  • Test if WDR73 has context-dependent functions based on cell cycle phase or differentiation state

  • Explore potential post-translational modifications that might alter WDR73 function

How can integrative multi-omics approaches incorporating WDR73 antibody-based techniques advance our understanding of rare genetic disorders?

Multi-omics strategies incorporating WDR73 antibody techniques can provide comprehensive insights:

• Integrated experimental design:

  • Chromatin immunoprecipitation followed by sequencing (ChIP-seq) to identify WDR73-associated genomic regions

  • RNA immunoprecipitation (RIP) to identify WDR73-associated RNAs

  • Proteomics using WDR73 antibodies for immunoprecipitation followed by mass spectrometry

  • Transcriptomics (RNA-seq) to identify differentially expressed genes in WDR73-deficient models

• Computational integration frameworks:

  • Correlate WDR73 binding patterns with transcriptional changes

  • Map protein-protein interactions to pathway perturbations

  • Identify network nodes most susceptible to therapeutic intervention

• Patient-derived models:

  • Apply multi-omics approaches to patient-derived cells (fibroblasts, iPSCs, differentiated podocytes)

  • Compare with engineered cell lines carrying identical mutations

  • Correlate molecular findings with clinical phenotypes

• Technical considerations for antibody-based multi-omics:

  • Validate antibody specificity for each application (ChIP, IP, RIP)

  • Use spike-in controls and appropriate normalization strategies

  • Consider epitope accessibility in different experimental contexts

By implementing these integrative approaches, researchers can move beyond isolated findings to develop comprehensive models of how WDR73 dysfunction leads to the complex phenotypes observed in Galloway-Mowat syndrome and related disorders.