wdr81 Antibody

What is WDR81 and why is it significant for neurological research?

WDR81 (WD repeat domain 81) is a 1941 amino acid protein with a molecular mass of approximately 212 kDa that contains one BEACH domain and five WD repeats . It has significant research importance due to its association with cerebellar ataxia, mental retardation, and quadrupedal locomotion syndrome (CAMRQ2) . WDR81 functions as a negative regulator of PI3 kinase/PI3K activity associated with endosomal membranes via BECN1, a core subunit of the PI3K complex . Recent studies have revealed its crucial role in aggrephagy (autophagic clearance of protein aggregates), making it particularly relevant for research on neurodegenerative diseases including Huntington's disease, Parkinson's disease, and Alzheimer's disease .

What is the subcellular localization pattern of WDR81 protein?

WDR81 demonstrates complex subcellular localization patterns across different cell types. Immunohistochemical and electron microscopy studies have shown that WDR81 localizes to:

• Membrane structures

• Cytoplasmic vesicles

• Mitochondria (particularly in Purkinje cell dendrites)

• Lysosomes

• Cytoplasm

Immunoelectron microscopy and subcellular fractionation analyses of cerebellum have confirmed the presence of WDR81 in mitochondria-enriched fractions (COX IV positive), particularly in Purkinje cells . Additionally, WDR81 has been found to associate with ubiquitin-positive protein foci, suggesting its involvement in protein quality control mechanisms .

What isoforms of WDR81 exist and how are they detected by antibodies?

Multiple WDR81 isoforms have been identified with distinct molecular weights:

Western blot analysis typically detects two bands of approximately 90 kDa and 80 kDa in size in cerebellum, brain, and spinal cord extracts . The 90 kDa band likely corresponds to isoform 2 (predicted ~96 kDa), while the 80 kDa band may correspond to isoforms 3 and/or 4. Interestingly, the full-length isoform 1 (~211.7 kDa) is often difficult to detect in CNS tissues, suggesting it may undergo proteolytic processing or have limited expression .

What are the optimal applications and dilutions for WDR81 antibodies?

Based on validated antibody data, WDR81 antibodies can be applied in multiple experimental contexts with specific recommended dilutions:

For immunohistochemistry on mouse cerebellum tissue, antigen retrieval with TE buffer pH 9.0 is suggested, although citrate buffer pH 6.0 may also be effective as an alternative . It's important to note that optimal dilutions should be determined empirically for each specific application and sample type .

How can I verify the specificity of WDR81 antibodies in my experiments?

To ensure antibody specificity, multiple validation approaches should be employed:

• Peptide blocking: Preincubation of the WDR81 antibody with the immunization peptide should abolish detection on Western blots and in immunostaining . This was demonstrated in cerebellum, brain, and spinal cord extracts, as well as in cerebellum, brainstem, and retina sections.

• Heterologous expression systems: Transfect cells (e.g., HEK293) with a WDR81 expression construct and verify antibody recognition of the overexpressed protein. In validated experiments, WDR81 antibody successfully recognized a ~100 kDa band in HEK cells transfected to express mouse WDR81 isoform 2 .

• Knockout/knockdown validation: Compare antibody signal in WDR81 knockout (KO-81) or siRNA-treated cells versus controls. Specific antibodies should show reduced or absent signal in knockout/knockdown conditions .

• Cross-reactivity assessment: Test the antibody against related proteins to ensure it doesn't cross-react with other WD repeat domain-containing proteins.

What are the critical factors for successful Western blotting of WDR81?

Several technical considerations are crucial for reliable detection of WDR81 by Western blotting:

• Sample preparation: For optimal detection, prepare fresh lysates from tissues or cells using a buffer containing protease inhibitors to prevent degradation of WDR81.

• Protein loading: Due to the relatively low abundance of WDR81 in some tissues, load sufficient protein (30-50 μg) per lane.

• Protein transfer: Use a semi-dry or wet transfer system with methanol-containing buffer for efficient transfer of high molecular weight proteins.

• Antibody incubation: Overnight incubation at 4°C with primary antibody at the recommended dilution (1:500-1:1000) yields optimal results .

• Detection systems: Enhanced chemiluminescence (ECL) systems with longer exposure times may be necessary to visualize less abundant isoforms.

• Observed molecular weights: Expect to detect bands at 74-80 kDa and/or 250 kDa depending on the tissue and antibody used .

How can WDR81 antibodies be used to study neurodegenerative disease mechanisms?

WDR81 antibodies are powerful tools for investigating neurodegenerative disease mechanisms:

• Protein aggregate association: WDR81 associates with ubiquitin-positive protein aggregates in various neurodegenerative disease models. Immunofluorescence co-staining with WDR81 antibodies and ubiquitin antibodies can visualize this association .

• Autophagy pathway analysis: WDR81 interacts with p62 and LC3C, key components of the autophagy pathway. Co-immunoprecipitation experiments using WDR81 antibodies can characterize these interactions and their disruption in disease states .

• Expression level changes: In hippocampus and cortex of patients with Huntington's disease, Parkinson's disease, and Alzheimer's disease, protein levels of endogenous WDR81 are decreased while p62 accumulates significantly. Western blotting with WDR81 antibodies can quantify these changes .

• Therapeutic target validation: Overexpression of WDR81 restores the viability of fibroblasts from Huntington's disease patients, suggesting therapeutic potential. WDR81 antibodies can confirm overexpression and localization in these rescue experiments .

What approaches can resolve conflicting WDR81 subcellular localization data?

Conflicting data regarding WDR81 subcellular localization can be resolved through:

• Super-resolution microscopy: Techniques like STORM or STED microscopy provide nanometer-scale resolution to precisely locate WDR81 in cellular compartments.

• Organelle fractionation: Differential centrifugation to isolate mitochondria, endosomes, and other organelles followed by Western blotting can quantitatively determine the distribution of WDR81 across subcellular compartments. This approach has successfully demonstrated WDR81's presence in mitochondrial-enriched fractions .

• Immunoelectron microscopy: Gold-labeled WDR81 antibodies can directly visualize protein localization at the ultrastructural level, as demonstrated in studies showing WDR81 in dendritic mitochondria of Purkinje cells .

• Domain-specific antibodies: Using antibodies targeting different domains of WDR81 can help determine if specific protein regions localize to different subcellular compartments.

• Live-cell imaging: Fluorescently tagged WDR81 combined with organelle markers can track dynamic localization patterns.

How do disease-associated mutations affect WDR81 detection by antibodies?

Disease-associated mutations in WDR81 may influence antibody detection in complex ways:

How can I troubleshoot weak or absent WDR81 signal in immunohistochemistry?

When encountering weak or absent WDR81 signal in immunohistochemistry:

• Antigen retrieval optimization: Test multiple antigen retrieval methods. For WDR81, TE buffer pH 9.0 is recommended, but citrate buffer pH 6.0 may be an effective alternative .

• Fixation conditions: Overfixation can mask epitopes. Test reduced fixation times or alternative fixatives (4% PFA or -20°C ethanol have been validated for WDR81 immunofluorescence) .

• Antibody concentration: Increase primary antibody concentration or incubation time. For WDR81, try 1:200 dilution instead of 1:800 .

• Signal amplification: Implement tyramide signal amplification (TSA) or other amplification systems for low-abundance targets.

• Detection system sensitivity: Switch to a more sensitive detection system, such as polymer-based systems instead of ABC methods.

• Tissue-specific considerations: WDR81 is highly expressed in Purkinje cells of the cerebellum and photoreceptor cells, making these tissues ideal positive controls .

What controls are essential when using WDR81 antibodies for critical research?

Essential controls for WDR81 antibody experiments include:

• Peptide blocking control: Pre-incubate antibody with immunizing peptide to demonstrate binding specificity .

• Positive tissue controls: Include cerebellum sections (where WDR81 is expressed in Purkinje cells) as a positive control .

• Negative controls:

  • Primary antibody omission

  • Isotype control antibody

  • Tissues from WDR81 knockout animals when available

• Loading controls: For Western blots, include housekeeping proteins like GAPDH or α-actin to normalize WDR81 expression levels .

• Molecular weight markers: To correctly identify WDR81 isoforms at 74-80 kDa and/or 250 kDa .

• Cross-validation: Confirm findings using multiple antibodies targeting different epitopes of WDR81 when possible.

How does sample preparation affect WDR81 detection in different applications?

Sample preparation significantly impacts WDR81 detection across applications:

• Western blotting:

  • Lysis buffer composition affects extraction efficiency (RIPA buffer with protease inhibitors is recommended)

  • Denaturing conditions influence antibody epitope accessibility

  • Fresh samples yield better results than frozen samples for some isoforms

• Immunohistochemistry/Immunofluorescence:

  • Fixation method impacts epitope preservation (4% PFA fixation or -20°C ethanol fixation have been validated)

  • Section thickness (optimal: 5-10 μm) affects antibody penetration

  • Antigen retrieval method is crucial (TE buffer pH 9.0 recommended)

• Electron microscopy:

  • Embedding medium affects antibody accessibility

  • Fixation protocols must balance ultrastructure preservation with epitope retention

  • Gold particle size selection impacts resolution and sensitivity

• Cell types and tissues:

  • WDR81 expression varies across tissues, with highest levels in cerebellum, brain, and spinal cord

  • Cell-specific expression patterns (e.g., Purkinje cells, photoreceptors) should guide experimental design

How should WDR81 antibodies be integrated into autophagy pathway analysis?

For comprehensive autophagy pathway analysis with WDR81 antibodies:

• Co-localization studies: Combine WDR81 antibodies with markers for:

  • Ubiquitinated proteins (WDR81 associates with ubiquitin-positive protein foci)

  • p62/SQSTM1 (WDR81 interacts directly with p62)

  • LC3 isoforms (especially LC3C, which preferentially interacts with WDR81)

  • Autophagosomes and autolysosomes

• Functional assays:

  • Monitor autophagic flux using bafilomycin A1 treatment while tracking WDR81 and LC3 levels

  • Assess cargo clearance rates in WDR81 wildtype versus knockdown/knockout conditions

• Domain-specific interactions:

  • WDR81's BEACH domain contains canonical LC3-interacting regions critical for LC3C recruitment

  • The WD40 repeats are essential for WDR81 interaction with covalently bound ATG5-ATG12

• Experimental manipulations:

  • Induce protein aggregation with MG132 (proteasome inhibitor) or puromycin to trigger WDR81 recruitment to ubiquitin-positive foci

  • Avoid non-specific stress inducers like tunicamycin or FCCP, which don't trigger WDR81 focal structures

What are the best experimental models for studying WDR81 function using antibodies?

Optimal experimental models for WDR81 research include:

• Cellular models:

  • HeLa cells (validated for WDR81 expression and knockdown/knockout studies)

  • SH-SY5Y neuroblastoma cells (relevant for neuronal disease mechanisms)

  • Primary neurons (especially cerebellar Purkinje cells)

  • Patient-derived fibroblasts (particularly from HD patients for rescue experiments)

• Animal models:

  • nur5 mouse model (carries WDR81 L1349P mutation, displays cerebellar ataxia and retinal degeneration)

  • WDR81 BAC transgenic mice (for rescue experiments)

  • C. elegans SORF-2 deletion model (homolog of WDR81, shows p62 body accumulation)

• Disease models:

  • Huntington's disease models (polyQ expansion constructs)

  • Parkinson's disease models (α-synuclein overexpression)

  • Alzheimer's disease models (examining WDR81 levels in affected tissues)

• Tissue samples:

  • Human brain samples from neurodegenerative disease patients (for examining WDR81 expression changes)

  • Cerebellum sections (for studying WDR81 in Purkinje cells)

  • Retinal sections (for examining WDR81 in photoreceptors)

How can WDR81 antibodies contribute to therapeutic development for neurodegenerative diseases?

WDR81 antibodies can facilitate therapeutic development through:

• Target validation: Confirm WDR81's reduced expression in patient samples from Huntington's, Parkinson's, and Alzheimer's diseases using immunohistochemistry and Western blotting .

• Mechanism characterization: Elucidate how WDR81 facilitates the recruitment of autophagic proteins onto protein aggregates (e.g., Htt polyQ aggregates) using co-immunoprecipitation and immunofluorescence .

• Domain-function relationships: Determine which WDR81 domains are critical for therapeutic function:

  • BEACH and MFS domains (sufficient for recruitment onto Htt polyQ aggregates)

  • WD40 repeats (essential for interaction with ATG5-ATG12)

• Therapeutic screening: Develop high-content screening assays using WDR81 antibodies to identify compounds that:

  • Enhance WDR81 expression

  • Promote WDR81 recruitment to protein aggregates

  • Strengthen WDR81 interactions with autophagy machinery

• Efficacy assessment: Monitor restoration of autophagic clearance in disease models following therapeutic intervention by tracking:

  • WDR81 levels and localization

  • p62 accumulation (which occurs when WDR81 function is compromised)

  • Aggregate clearance efficiency