WDR46 Antibody

What is WDR46 and why is it significant for immunological research?

WDR46 (also known as C6orf11, BING4, FP221, and UTP7) is a WD repeat-containing protein located on chromosome 6p21.3 at the centromere side of the class II major histocompatibility complex region. This 610 amino acid protein contains G-beta (WD40) repeats found in the β-subunits of G-proteins, a class I type aminotransferase pyridoxal phosphate attachment site motif, and a class I aminoacyl-tRNA synthetase adenylate binding site motif .

The significance of WDR46 in immunological research stems from its association with aspirin-exacerbated respiratory disease (AERD) and respiratory function. Studies have demonstrated that WDR46 variants are significantly associated with both AERD risk and forced expiratory volume in the first second (FEV1) decline after aspirin provocation in asthma patients . WDR46 also functions as a scaffold component of the nucleolar structure and is required for the localization of DDX21 and NCL to the granular compartment of the nucleolus .

What applications are most suitable for WDR46 antibodies?

WDR46 antibodies have been validated for multiple experimental applications:

When selecting an application, it's recommended that antibodies be titrated in each testing system to obtain optimal results as performance can be sample-dependent .

What are the optimal storage conditions for WDR46 antibodies?

Most commercial WDR46 antibodies should be stored at -20°C for long-term stability. The typical storage buffer consists of PBS (pH 7.2) with 0.02% sodium azide and 40-50% glycerol . Under these conditions, antibodies remain stable for approximately one year after shipment.

For -20°C storage, aliquoting is generally unnecessary for small volume products (like 25 μl sizes), though some preparations may contain 0.1% BSA as a stabilizer . Always avoid repeated freeze-thaw cycles which can compromise antibody activity and specificity.

What is the recommended Western blot protocol for WDR46 antibody?

For optimal Western blot results with WDR46 antibody, follow this methodological approach:

• Sample Preparation: Prepare cell or tissue lysates using standard protocols. HeLa cells, Jurkat cells, and spleen tissue from mouse or rat have shown good detection of WDR46 .

• Protein Loading: Load 20-40 μg of total protein per lane.

• Gel Electrophoresis: Use 8-10% SDS-PAGE as WDR46 has an observed molecular weight of 70-75 kDa (calculated molecular weight is 68 kDa) .

• Transfer: Transfer proteins to PVDF or nitrocellulose membrane using standard transfer conditions.

• Blocking: Block in 5% non-fat milk or BSA in TBST for 1 hour at room temperature.

• Primary Antibody Incubation: Dilute WDR46 antibody 1:2000-1:10000 in blocking buffer and incubate overnight at 4°C .

• Washing: Wash 3-5 times with TBST.

• Secondary Antibody: Apply appropriate HRP-conjugated secondary antibody and develop using chemiluminescence.

When interpreting results, expect to see a main band at 70-75 kDa representing WDR46 .

How can immunohistochemistry protocols be optimized for WDR46 detection?

For successful immunohistochemistry with WDR46 antibody in paraffin-embedded tissues:

• Deparaffinization and Rehydration: Process sections through xylene and graded alcohols.

• Antigen Retrieval: This step is critical as fixation can mask epitopes. Heat-induced epitope retrieval (HIER) in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) is typically recommended .

• Blocking: Block endogenous peroxidase activity with 3% H₂O₂ and non-specific binding with 5-10% normal serum.

• Primary Antibody: Dilute WDR46 antibody 1:20-1:50 and incubate sections overnight at 4°C or for 1-2 hours at room temperature .

• Detection System: Use an appropriate detection system based on your primary antibody species (rabbit for most WDR46 antibodies).

• Counterstaining: Lightly counterstain with hematoxylin.

• Positive Controls: Include spleen tissue which has demonstrated reliable WDR46 expression .

• Negative Controls: Include sections with secondary antibody only or with a non-specific primary antibody of the same isotype.

What considerations are important for using WDR46 antibodies in flow cytometry?

When using WDR46 antibodies for intracellular flow cytometry:

• Sample Preparation: Use at least 10⁶ cells per sample. Jurkat cells have been validated for positive detection .

• Fixation and Permeabilization: Since WDR46 is an intracellular protein, proper fixation and permeabilization are essential. Use 4% paraformaldehyde for fixation followed by permeabilization with 0.1-0.5% saponin or 0.1% Triton X-100.

• Antibody Concentration: Use 0.25 μg of WDR46 antibody per 10⁶ cells in a 100 μl suspension .

• Controls: Include isotype control to establish background staining levels.

• Gating Strategy: Consider a hierarchical gating strategy starting with intact cells (FSC vs SSC), followed by singlets, and then your population of interest.

• Analysis: Because WDR46 has relatively uniform expression across many cell types, look for shifts in mean fluorescence intensity rather than discrete positive/negative populations.

How can WDR46 antibodies be used to study aspirin-exacerbated respiratory disease (AERD)?

WDR46 has been identified as a genetic risk factor for AERD, with significant associations between WDR46 variants and both disease risk and FEV1 decline after aspirin provocation . Researchers can use WDR46 antibodies in several methodological approaches to study AERD:

• Expression Analysis: Compare WDR46 protein expression levels between AERD patients and controls using Western blot or immunohistochemistry on airway biopsies.

• Single-Nucleotide Polymorphism (SNP) Effects: Investigate how specific SNPs (particularly rs463260, rs446735, rs455567, and rs469064) affect protein expression or function. These SNPs have shown significant association with AERD risk (P=0.007-0.04) and FEV1 decline (P=0.006-0.03) .

• Cell Type-Specific Expression: Use immunofluorescence or flow cytometry to determine if WDR46 expression differs between cell types in AERD vs. non-AERD patients.

• Functional Studies: Employ co-immunoprecipitation with WDR46 antibodies to identify potential protein-protein interactions that may be altered in AERD.

• Pathway Analysis: Use WDR46 antibodies in combination with antibodies against other proteins in relevant inflammatory pathways to understand the mechanistic role of WDR46 in aspirin-induced inflammation.

The logistic regression analysis below summarizes the significant association between WDR46 variants and AERD:

Table adapted from the association analysis between WDR46 variants and AERD risk

What are effective methods for validating WDR46 antibody specificity?

Validating antibody specificity is critical for experimental rigor. For WDR46 antibodies, consider these methodological approaches:

• Protein Array Validation: Some WDR46 antibodies have been validated against protein arrays containing the target protein plus 383 other non-specific proteins . This approach can identify potential cross-reactivity issues.

• Knockout/Knockdown Controls:

  • Use siRNA knockdown of WDR46 to demonstrate decreased signal in Western blot or immunostaining

  • If available, use CRISPR/Cas9-generated knockout cell lines as negative controls

• Epitope Blocking: Pre-incubate the WDR46 antibody with a recombinant protein control fragment (such as Human WDR46 aa 395-475) at a 100x molar excess for 30 minutes at room temperature before application to samples . This should abolish specific staining.

• Multiple Antibodies Approach: Use two independent antibodies targeting different epitopes of WDR46 and compare staining patterns. Concordant results increase confidence in specificity.

• Recombinant Expression: Overexpress WDR46 in a cell line with low endogenous expression and confirm increased signal.

• Mass Spectrometry: Perform immunoprecipitation with the WDR46 antibody followed by mass spectrometry to confirm the identity of the precipitated protein.

How can researchers investigate the function of WDR46 in nucleolar structure using antibodies?

WDR46 serves as a scaffold component of the nucleolar structure and is required for localizing DDX21 and NCL to the granular compartment of the nucleolus . To investigate this function:

• Co-localization Studies: Perform dual immunofluorescence with antibodies against WDR46 and nucleolar markers (fibrillarin, nucleolin, DDX21) to examine their spatial relationships.

• Protein-Protein Interaction Analysis:

  • Use WDR46 antibodies for co-immunoprecipitation experiments to identify interaction partners

  • For co-IP, use 0.5-4.0 μg of WDR46 antibody for 1.0-3.0 mg of total protein lysate

  • Analyze precipitates by Western blot or mass spectrometry

• Nucleolar Disruption Experiments: Treat cells with RNA polymerase I inhibitors (e.g., actinomycin D) to disrupt nucleolar structure and observe changes in WDR46 localization using immunofluorescence.

• Proximity Ligation Assays (PLA): Use WDR46 antibody paired with antibodies against potential interacting partners to visualize and quantify protein-protein interactions in situ.

• ChIP Sequencing: If WDR46 associates with chromatin, perform ChIP-seq using WDR46 antibodies to identify genomic binding sites.

• Ultrastructural Analysis: Use immunogold labeling with WDR46 antibodies for electron microscopy to precisely localize WDR46 within nucleolar subcompartments.

How can inconsistent WDR46 antibody staining be resolved in immunohistochemistry?

Inconsistent staining with WDR46 antibodies in IHC can be addressed through these methodological approaches:

• Antigen Retrieval Optimization: Test different antigen retrieval methods:

  • Citrate buffer (pH 6.0) vs. EDTA buffer (pH 9.0)

  • Varying retrieval times (10-30 minutes)

  • Pressure cooker vs. microwave heating

• Antibody Titration: Test a range of dilutions around the recommended 1:20-1:50 dilution to find the optimal concentration for your specific tissue.

• Incubation Conditions: Compare overnight incubation at 4°C vs. shorter incubations (1-2 hours) at room temperature.

• Signal Amplification: Consider using polymer-based detection systems or tyramide signal amplification for weak signals.

• Tissue Processing: Optimize fixation time for fresh samples; for archived samples, explore additional permeabilization steps.

• Blocking Optimization: Increase blocking time or concentration of blocking agents to reduce background.

• Positive Controls: Include tissues known to express WDR46 (such as spleen) in each staining run to confirm antibody functionality .

• Antibody Quality: Confirm antibody hasn't degraded and consider testing a new lot or a different clone if problems persist.

What are the potential causes and solutions for multiple bands in WDR46 Western blots?

When multiple bands appear in Western blots with WDR46 antibodies, consider these potential causes and solutions:

• Protein Degradation:

  • Add fresh protease inhibitors to lysis buffer

  • Keep samples cold during preparation

  • Reduce sample handling time

• Post-translational Modifications:

  • WDR46 may exist in different phosphorylation states

  • Treat samples with phosphatases to determine if higher molecular weight bands are phosphorylated forms

• Splice Variants:

  • WDR46 has multiple transcript variants that could produce proteins of different sizes

  • Compare band patterns with information on known splice variants

• Non-specific Binding:

  • Increase blocking time or concentration

  • Try different blocking agents (milk vs. BSA)

  • Increase washing stringency

  • Optimize antibody dilution (try higher dilutions like 1:10000)

• Cross-reactivity:

  • Perform peptide competition assays with recombinant WDR46 protein fragments

  • Consider using a more specific antibody or one targeting a different epitope

• Sample Preparation:

  • Ensure complete denaturation of proteins by adequate heating in sample buffer

  • Check for appropriate reducing conditions

The expected molecular weight for WDR46 is 68 kDa (calculated) with observed migration between 70-75 kDa .

How should WDR46 antibodies be applied in studying genetic variants and SNPs?

When using WDR46 antibodies to study genetic variants and SNPs, particularly those associated with AERD like rs463260, rs446735, rs455567, and rs469064 , implement these methodological approaches:

• Genotype-Phenotype Correlation:

  • Genotype samples for the SNPs of interest

  • Use WDR46 antibodies in Western blot or IHC to quantify protein expression

  • Correlate genotypes with protein expression levels

• Allele-Specific Effects:

  • For SNPs in coding regions, determine if they affect epitope recognition

  • For the rs463260 G>A variant in the 5' UTR, which potentially affects an upstream open reading frame (uORF) , measure protein levels in cells with different genotypes

• Functional Analysis:

  • Generate cell lines with specific WDR46 variants using CRISPR/Cas9

  • Compare protein expression, localization, and function using WDR46 antibodies

• Tissue-Specific Effects:

  • Analyze WDR46 expression in relevant tissues (lung, airway epithelium) from individuals with different genotypes

  • Compare expression levels in health vs. disease states

• Interaction Studies:

  • Investigate if SNPs affect protein-protein interactions by comparing co-immunoprecipitation results across genotypes

  • Use proximity ligation assays to quantify differences in protein interactions in situ

Consider that the rs463260 polymorphism showed the most robust association with AERD risk (P=0.007, P corrected=0.01) and FEV1 decline (P=0.005, P corrected=0.01) in a co-dominant genetic model .

How can WDR46 antibodies contribute to understanding small subunit processome function?

WDR46 (also known as UTP7) plays a role in the small subunit processome, which is involved in ribosome biogenesis. Researchers can use WDR46 antibodies to investigate this function through:

• Nucleolar Proteomics:

  • Immunoprecipitate WDR46 from nucleolar extracts

  • Identify associated proteins by mass spectrometry

  • Map the interaction network within the processome

• RNA-Protein Interactions:

  • Perform RNA immunoprecipitation (RIP) using WDR46 antibodies

  • Identify associated pre-rRNAs and snoRNAs

  • Determine binding sites using CLIP-seq methods

• Functional Disruption Analysis:

  • Deplete WDR46 using siRNA or CRISPR

  • Use WDR46 antibodies to confirm knockdown efficiency

  • Analyze effects on processome assembly and pre-rRNA processing

• Developmental Biology:

  • Study the role of WDR46 in pluripotent stem cells, as referenced in publications using WDR46 antibodies

  • Investigate changes in expression and localization during differentiation

  • Correlate with ribosome biogenesis in developmental contexts

• Disease Models:

  • Apply WDR46 antibodies to study ribosomopathies

  • Investigate nucleolar stress responses

  • Explore potential connections between ribosome biogenesis defects and inflammatory conditions like AERD

What are the latest methodological approaches for studying WDR46 in immune cell function?

Based on WDR46's location in the MHC region and its association with inflammatory conditions, researchers can employ these advanced methodological approaches using WDR46 antibodies:

• Single-Cell Analysis:

  • Perform high-dimensional flow cytometry or mass cytometry (CyTOF) with WDR46 antibodies

  • Correlate WDR46 expression with immune cell activation states

  • Identify cell subsets with differential WDR46 expression

• Spatial Transcriptomics and Proteomics:

  • Combine WDR46 immunofluorescence with in situ RNA sequencing

  • Map WDR46 protein expression in tissue microenvironments

  • Correlate with cellular identities and functional states

• Immune Cell Signaling:

  • Use phospho-flow cytometry with WDR46 antibodies

  • Investigate connections between WDR46 and immune signaling pathways

  • Study dynamics of WDR46 expression during immune cell activation

• Systems Immunology:

  • Incorporate WDR46 expression data into computational models of immune function

  • Identify regulatory networks involving WDR46

  • Predict functional consequences of WDR46 variants

• Organoid and 3D Culture Systems:

  • Apply WDR46 antibodies in immunostaining of airway epithelial organoids

  • Study WDR46 expression in response to inflammatory stimuli

  • Model AERD pathophysiology in differentiated cultures

The Human Protein Atlas categorizes WDR46 expression patterns across immune cells, making it possible to target specific immune cell populations for functional studies .