WDR62 Antibody

What is WDR62 and why is it important in neurodevelopmental research?

WDR62 (WD repeat domain 62) is a scaffold protein critical for cerebral cortical development. In humans, the canonical protein has 1518 amino acid residues with a molecular mass of approximately 166 kDa . Its importance stems from several key functions:

• Required for proper cerebral cortical development

• Plays essential roles in neuronal proliferation and migration

• Functions in mother-centriole-dependent centriole duplication

• Mutations in WDR62 are the second most common cause of autosomal recessive primary microcephaly (MCPH)

• Acts as a scaffold protein that recruits components of the JNK signaling pathway

The protein's significance in brain development makes WDR62 antibodies invaluable tools for studying neurodevelopmental disorders, particularly microcephaly and related conditions affecting brain size and architecture.

What are the common applications for WDR62 antibodies in research?

WDR62 antibodies are employed in multiple experimental techniques:

These applications allow researchers to study WDR62 expression, localization, and interactions in various experimental contexts .

What is the subcellular localization of WDR62 and how does this impact antibody selection?

WDR62 exhibits dynamic subcellular localization that changes during the cell cycle:

• Primarily localizes to the Golgi apparatus during interphase in cultured cells and human fetal brain tissue

• Translocates to the mitotic spindle poles during cell division in a microtubule-dependent manner

• Can be found in both nuclear and cytoplasmic compartments

This dynamic localization has important implications for antibody selection:

• For Golgi localization studies, co-staining with Golgi markers like GOLGA1 or GOLGA2 is recommended

• For mitotic spindle studies, antibodies recognizing epitopes not obscured during mitotic complex formation should be selected

• Different fixation methods may preserve different localizations (e.g., paraformaldehyde for spindle pole localization)

Understanding this localization pattern is crucial for experimental design and interpretation of results.

How can researchers validate the specificity of WDR62 antibodies?

Validating WDR62 antibody specificity is crucial for reliable results. Recommended validation approaches include:

• siRNA knockdown validation:

  • Transfect cells with WDR62-specific siRNA

  • No signal should be detected in WDR62-silenced cells by immunostaining

  • Include transfection controls (e.g., LMNA-Cy3) to confirm transfection efficiency

• Recombinant expression systems:

  • Express full-length human WDR62 cDNA with epitope tags (e.g., HA tag)

  • Perform parallel staining with tag-specific and WDR62-specific antibodies

  • Signal co-localization confirms specificity

• Multiple antibody approach:

  • Use antibodies targeting different epitopes of WDR62

  • Consistent localization patterns across antibodies suggest specificity

  • Consider antibodies raised against different regions (N-terminal vs. C-terminal)

• Western blot analysis:

  • Confirm single band at expected molecular weight (~166 kDa)

  • Test across multiple cell lines (e.g., HeLa, HEK-293T)

  • Include concentration gradients to demonstrate specificity

What are the optimal protocols for detecting WDR62 in different subcellular compartments?

Detecting WDR62 in different subcellular locations requires optimized protocols:

For Golgi apparatus localization:

• Fix cells with 4% paraformaldehyde for 10-15 minutes

• Co-stain with established Golgi markers (GOLGA1, GOLGA2/GM130)

• To confirm Golgi association, treat cells with nocodazole (3 hours) to fragment the Golgi

• Approximately 58% of Golgi ministacks will remain WDR62-positive after fragmentation

For mitotic spindle pole localization:

• Synchronize cells to enrich for mitotic populations

• For microtubule dependency studies, treat with microtubule-disrupting agents

• Use metaphase cells for optimal spindle pole visualization

• Co-stain with centrosomal markers (e.g., CDK5RAP2) to confirm localization

For interaction studies:

• WDR62 interacts with CDK5RAP2, AURKA, and TPX2

• Co-immunoprecipitation can detect these interactions

• Even truncated forms (e.g., D955AfsX112 mutation) maintain some interaction capacity

How do mutations in WDR62 affect antibody binding and experimental outcomes?

WDR62 mutations have significant implications for antibody binding and experimental design:

• Epitope accessibility: Mutations may alter protein folding, potentially masking or exposing different epitopes

• Truncation effects: C-terminal truncating mutations (e.g., D955AfsX112) can eliminate epitopes in that region

• Localization changes: Mutations may affect subcellular localization without preventing protein-protein interactions

• Expression levels: Some mutations can decrease protein stability, resulting in lower detection signals

For studies involving mutant forms:

• Use antibodies targeting epitopes outside the mutated region

• Perform parallel studies with tagged wild-type and mutant constructs

• Consider using antibodies against interaction partners as indirect readouts

• Validate antibody binding to the specific mutant variant before conducting extensive experiments

What are the best practices for WDR62 antibody-based studies in brain development and microcephaly research?

For brain development and microcephaly research:

• Model system selection:

  • iPSC-derived neural stem cells from patients with WDR62 mutations provide valuable disease models

  • Cerebral organoids can recapitulate 3D architecture affected in microcephaly

  • Consider both 2D and 3D models for comprehensive analysis

• Developmental timing:

  • WDR62 expression and localization change during neurodevelopment

  • Study multiple time points during differentiation protocols

  • Compare with appropriate developmental controls

• Co-analysis approaches:

  • Combine antibody staining with cell cycle markers

  • Assess both proliferation and differentiation markers

  • Evaluate spindle orientation in neural progenitors

• Mutation-specific considerations:

  • Different WDR62 mutations may have distinct effects on protein function and localization

  • Use isogenic corrected cell lines as controls for patient-derived cells

  • Consider antibodies targeting specific mutant forms when available

What technical challenges exist in optimizing Western blot protocols for WDR62 detection?

WDR62 Western blot optimization faces several technical challenges:

• High molecular weight considerations:

  • At 166 kDa, WDR62 requires extended transfer times or specialized protocols

  • Use low percentage gels (6-8%) for better resolution

  • Consider graduated transfer buffers with increasing methanol concentrations

• Loading and signal optimization:

  • Recommended protein loading: 5-50 μg total protein per lane

  • Optimal antibody concentration: ~0.04 μg/mL for sensitive detection

  • ECL detection with 30-second exposure provides clear bands in most cell types

• Isoform detection:

  • Up to 4 different isoforms have been reported

  • Use antibodies targeting conserved regions to detect multiple isoforms

  • Higher resolution gels may be needed to distinguish closely migrating isoforms

• Validation approach:

  • Test multiple cell lines (HeLa, HEK-293T recommended)

  • Include concentration gradients of total protein (5, 15, 50 μg)

  • Use positive controls with known WDR62 expression

How can researchers establish effective immunoprecipitation protocols for studying WDR62 protein interactions?

Effective immunoprecipitation (IP) of WDR62 requires careful optimization:

• Antibody selection and amount:

  • Use 0.5-4.0 μg of antibody per 1-3 mg of total protein lysate

  • Affinity-purified antibodies typically yield cleaner results

  • Consider rabbit polyclonal antibodies for higher avidity binding

• Lysis conditions:

  • Use buffers that preserve relevant protein interactions

  • For centrosomal protein interactions, include phosphatase inhibitors

  • For Golgi-related studies, avoid detergents that disrupt membrane integrity

• Interaction validation approach:

  • Reciprocal IP (pull down with partner antibody)

  • Overexpression studies with tagged constructs can confirm interactions

  • Known interaction partners include CDK5RAP2, AURKA, and TPX2

• Controls:

  • IgG control from same species as primary antibody

  • Input sample (pre-IP lysate) at 5-10% of IP amount

  • When available, lysate from WDR62-depleted cells as negative control

What considerations are important when using WDR62 antibodies for high-resolution imaging techniques?

For high-resolution imaging of WDR62:

• Fixation optimization:

  • Paraformaldehyde (4%) preserves most epitopes while maintaining structure

  • Cold methanol may better preserve centrosomal structures

  • Avoid harsh fixatives that may destroy epitopes

• Signal amplification strategies:

  • Consider fluorophore-conjugated secondary antibodies with high quantum yield

  • Tyramide signal amplification for weak signals

  • Avoid protocols that increase background (excessive amplification)

• Co-localization studies:

  • With Golgi markers: GOLGA1, GOLGA2/GM130

  • With centrosomal proteins: CDK5RAP2, CEP152, CEP63

  • With mitotic spindle markers: AURKA, TPX2

• Resolution considerations:

  • Super-resolution techniques (STED, STORM, PALM) can resolve centrosomal structures

  • For organoid work, clearing techniques may improve imaging depth

  • Z-stack acquisition with deconvolution improves resolution of subcellular structures

By implementing these optimized approaches, researchers can effectively utilize WDR62 antibodies to advance our understanding of neurodevelopmental disorders and fundamental cellular processes governed by this important protein.