WDR5 Recombinant Monoclonal Antibody

What is WDR5 and what are its primary cellular functions?

WDR5 functions as a critical component of protein complexes involved in epigenetic regulation, chromatin remodeling, and gene expression control. Its primary function is promoting histone methylation at specific gene promoters, thereby regulating gene activation and repression. WDR5 serves as a core subunit of all SET1/MLL histone methyltransferase complexes and is required for proper complex assembly and histone methyltransferase activity. It acts as an effector of histone H3 Lys4 methylation by recruiting SET1/MLL complexes to target loci and presenting the histone H3 amino-terminal tail for methylation. Proper WDR5-mediated regulation is crucial for normal development, cellular differentiation, and maintaining balanced gene expression in health and disease .

How are WDR5 recombinant monoclonal antibodies produced?

The production of WDR5 recombinant monoclonal antibodies begins with obtaining WDR5 antibody genes, which are then introduced into suitable host cells. These host cells are cultured for synthesizing WDR5 antibodies using a cell-based expression and translation system. This recombinant approach offers significant advantages, including improved purity and stability of the resulting antibodies, as well as enhanced affinity and specificity. Following synthesis, the antibodies undergo purification through affinity chromatography to isolate the target-specific antibodies. Quality control testing is then performed using various assays including ELISA, immunohistochemistry (IHC), and flow cytometry (FC) to ensure specificity for the human WDR5 protein .

What are the standard applications for WDR5 recombinant monoclonal antibodies?

WDR5 recombinant monoclonal antibodies can be utilized in multiple research applications:

These applications allow researchers to investigate WDR5 protein expression, localization, and interactions with chromatin and other proteins .

How should I optimize immunohistochemistry protocols using WDR5 antibodies?

For optimal immunohistochemistry using WDR5 antibodies, follow these methodological steps:

• Tissue preparation: Use paraffin-embedded tissue sections, performing proper dewaxing and hydration procedures.

• Antigen retrieval: Implement high-pressure antigen retrieval in citrate buffer (pH 6.0) to expose epitopes masked during fixation.

• Blocking: Block non-specific binding with 10% normal goat serum for 30 minutes at room temperature.

• Primary antibody incubation: Dilute WDR5 antibody to 1:50-1:200 in 1% BSA and incubate overnight at 4°C.

• Secondary antibody: Use an appropriate anti-rabbit polymer IgG labeled with HRP.

• Detection: Visualize with 0.79% DAB substrate and counterstain as needed.

The protocol can be performed on automated systems like the Leica Bond™ system for standardization. Always include positive and negative controls to validate staining specificity and troubleshoot protocol issues .

What are the recommended procedures for flow cytometry using WDR5 antibodies?

For flow cytometry applications with WDR5 antibodies, implement this methodology:

• Cell preparation: Fix cells in 4% formaldehyde and permeabilize with 0.2% TritonX-100 to allow antibody access to intracellular WDR5.

• Blocking: Block with 10% normal goat serum to reduce non-specific protein-protein interactions.

• Primary antibody incubation: Apply WDR5 antibody at a 1:50 dilution (approximately 1μg/1×10^6 cells) and incubate for 45 minutes at 4°C.

• Secondary antibody application: Use FITC-conjugated Goat Anti-rabbit IgG(H+L) at 1:200 dilution for 35 minutes at 4°C.

• Control samples: Include rabbit IgG (1μg/1×10^6 cells) under identical conditions as an isotype control.

• Data acquisition: Collect at least 10,000 events for reliable analysis.

This approach allows for quantitative assessment of WDR5 expression in different cell populations and following experimental manipulations .

What methodology should be employed for chromatin immunoprecipitation with WDR5 antibodies?

For chromatin immunoprecipitation (ChIP) using WDR5 antibodies:

• Sample preparation: Use approximately 4×10^6 cells and 10 μg of chromatin per immunoprecipitation.

• Antibody quantity: Use 10 μl of WDR5 antibody per ChIP reaction.

• Protocol optimization: Employ enzymatic chromatin shearing methods (such as those in SimpleChIP® Enzymatic Chromatin IP Kits) for optimal results.

• Controls: Include IgG control antibodies and positive control loci known to be bound by WDR5.

• Analysis: Analyze precipitated DNA using qPCR, microarrays (ChIP-chip), or sequencing (ChIP-seq).

For ChIP-seq applications, maintain the 1:50 dilution ratio and follow standard library preparation protocols. WDR5 ChIP-seq can identify genomic loci where WDR5 functions in histone methylation, particularly at promoters of ribosomal protein genes and other WDR5-regulated genes .

How can WDR5 antibodies be used to study the mechanism of WIN site inhibition?

WDR5 antibodies provide crucial tools for investigating WIN site inhibitor mechanisms:

• Chromatin displacement assays: Use ChIP or CUT&RUN with WDR5 antibodies before and after WIN site inhibitor treatment to quantify WDR5 displacement from chromatin. This approach has revealed that potent WIN site inhibitors cause rapid and comprehensive displacement of WDR5 from chromatin.

• Gene expression correlation: Combine ChIP-seq with RNA-seq to correlate changes in WDR5 chromatin occupancy with alterations in gene expression following inhibitor treatment. This methodology has demonstrated that WIN site inhibitors lead to commensurate decreases in expression of WDR5-bound genes, particularly ribosome protein genes.

• Mechanistic validation: Use WDR5 antibodies in combination with antibodies against H3K4 methylation marks to confirm that WIN site inhibition disrupts WDR5-dependent histone modifications.

• Protein interaction studies: Employ WDR5 antibodies in co-immunoprecipitation experiments to investigate how WIN site inhibitors affect WDR5 protein-protein interactions, especially with SET1/MLL complexes.

These approaches have helped establish that WIN site inhibitors function by displacing WDR5 from chromatin, leading to downstream effects on gene expression, translation, and cellular processes such as nucleolar stress and p53 induction .

What techniques can be combined with WDR5 antibodies to investigate its role in epigenetic regulation?

Several advanced techniques can be combined with WDR5 antibodies to investigate epigenetic regulation mechanisms:

• Sequential ChIP (ChIP-reChIP): Use WDR5 antibodies followed by antibodies against other epigenetic modifiers or histone marks to identify genomic regions where WDR5 co-localizes with specific epigenetic regulators.

• Proximity ligation assays: Combine WDR5 antibodies with antibodies against potential interaction partners to visualize and quantify protein-protein interactions in situ.

• CRISPR-based genomic editing: Use WDR5 antibodies to validate the effects of genomic modifications on WDR5 recruitment and function at specific loci.

• Mass spectrometry analysis: Use WDR5 antibodies for immunoprecipitation followed by mass spectrometry to identify novel WDR5-interacting proteins in different cellular contexts.

• Single-cell analysis: Employ WDR5 antibodies in single-cell analytical techniques to investigate cell-to-cell variability in WDR5 expression and localization.

These integrative approaches help elucidate WDR5's role in coordinating histone methylation with other epigenetic processes and understanding the consequences of disrupting these functions .

How does WDR5 contribute to cancer development and progression?

WDR5 plays significant roles in cancer through multiple mechanisms:

• Epigenetic dysregulation: WDR5's function in SET1/MLL histone methyltransferase complexes affects genome-wide H3K4 methylation patterns, potentially leading to aberrant gene expression in cancer cells. Proper WDR5-mediated regulation is crucial for maintaining the balance of gene expression in health and disease.

• Oncogenic partnerships: WDR5 serves as a co-factor for MYC, a well-established oncogene, contributing to its ability to promote malignant transformation and progression.

• Ribosomal protein gene regulation: WDR5 controls the expression of ribosome protein genes, and disruption of this function through WIN site inhibitors causes translational inhibition, nucleolar stress, and p53 induction.

• EMT promotion: WDR5 plays a critical role in promoting the epithelial-to-mesenchymal transition (EMT), a key process in cancer metastasis.

• Cancer-specific expression: Aberrant WDR5 expression has been implicated in various cancers, including leukemias, breast cancer, and bladder cancer.

These findings position WDR5 as a promising therapeutic target in numerous bloodborne and solid cancers, with particular interest in targeting the WIN site of WDR5 for pharmacological inhibition .

What methodologies can be used to study WDR5 in cancer models using WDR5 antibodies?

Researchers can employ several methodologies to study WDR5 in cancer models:

• Expression profiling: Use WDR5 antibodies in immunohistochemistry and Western blotting to analyze WDR5 expression levels across different cancer types, stages, and in comparison to normal tissues.

• Chromatin occupancy mapping: Apply ChIP-seq with WDR5 antibodies to identify cancer-specific WDR5 binding sites and associated genes, particularly focusing on ribosomal protein genes and MYC-regulated genes.

• Therapeutic response monitoring: Utilize WDR5 antibodies to evaluate changes in WDR5 expression, localization, and chromatin binding following treatment with epigenetic modulators or WIN site inhibitors.

• Functional studies: Combine WDR5 antibodies with genetic manipulation (knockdown/overexpression) to correlate WDR5 levels with cancer phenotypes like proliferation, apoptosis resistance, and metastatic potential.

• Patient-derived xenograft (PDX) models: Apply WDR5 antibodies to assess WDR5 expression and function in PDX models, which better recapitulate human tumor heterogeneity.

These approaches help characterize WDR5's contribution to cancer biology and identify contexts where targeting WDR5 might have therapeutic value .

How can I address weak or non-specific signals when using WDR5 antibodies?

When encountering weak or non-specific signals with WDR5 antibodies, implement these troubleshooting steps:

• Antibody titration: Optimize antibody concentration by testing a range of dilutions. Starting recommendations are 1:50-1:200 for IHC and FC, and 1:1000 for Western blotting.

• Antigen retrieval optimization: For IHC, test different antigen retrieval methods, focusing on citrate buffer (pH 6.0) with high-pressure treatment as recommended.

• Blocking conditions: Increase blocking time or use alternative blocking agents if background is high. The recommended protocol uses 10% normal goat serum.

• Incubation conditions: Adjust primary antibody incubation time and temperature. For IHC, overnight incubation at 4°C is recommended.

• Detection system sensitivity: Ensure the secondary antibody and detection system are compatible and sufficiently sensitive. For IHC, goat anti-rabbit polymer IgG labeled with HRP and visualization with 0.79% DAB is recommended.

• Positive and negative controls: Always include known positive samples and appropriate negative controls (including isotype controls for FC) to validate staining specificity.

• Sample preparation: Ensure proper fixation and processing of samples, as overfixation can mask epitopes while inadequate fixation may lead to poor morphology.

Systematic optimization of these parameters should improve signal-to-noise ratio and specificity when working with WDR5 antibodies .

What bioinformatic approaches help analyze WDR5 ChIP-seq data?

Analyzing WDR5 ChIP-seq data requires specialized bioinformatic approaches:

• Peak calling: Use algorithms like MACS2 to identify genomic regions with significant WDR5 binding. Parameters should be optimized based on control samples and known WDR5 binding sites.

• Genomic annotation: Map WDR5 binding sites to genomic features (promoters, enhancers, gene bodies) to understand the distribution of WDR5 across the genome.

• Motif analysis: Identify DNA sequence motifs enriched in WDR5 binding sites, which may reveal direct or indirect DNA binding preferences.

• Integration with histone modification data: Correlate WDR5 binding with H3K4 methylation patterns (particularly H3K4me3) and other histone modifications to understand functional relationships.

• Gene expression correlation: Integrate with RNA-seq data to identify genes whose expression correlates with WDR5 binding, focusing on ribosomal protein genes and other WDR5-regulated genes identified in previous studies.

• Comparative analysis: Compare WDR5 binding profiles before and after treatment with WIN site inhibitors to identify sites most sensitive to inhibition.

• Pathway enrichment analysis: Perform Gene Ontology and pathway analysis on WDR5-bound genes to identify biological processes regulated by WDR5.

These approaches help extract meaningful biological insights from complex ChIP-seq datasets and understand WDR5's role in chromatin biology .