wdr61 Antibody

What is the biological function of WDR61?

WDR61 serves as a subunit in two important protein complexes: the PAF complex (PAF1C) and the SKI complex. In the PAF complex, WDR61 contributes to transcriptional regulation and is implicated in the regulation of development and maintenance of embryonic stem cell pluripotency . As part of the SKI complex, WDR61 is involved in exosome-mediated RNA decay and associates with transcriptionally active genes in a manner dependent on PAF1C . Recent research has revealed that WDR61 interacts with R-loops, and its loss leads to R-loop accumulation in breast tumor cells, causing DNA damage and subsequent inhibition of cell proliferation .

What is the molecular structure and characteristics of WDR61?

WDR61 is a 34 kDa protein composed of 305 amino acids and contains WD-repeat domains, which typically form a beta-propeller structure that serves as a platform for protein-protein interactions . The protein is conserved across species, with reactive epitopes in human, mouse, and rat samples . When observed on Western blots, WDR61 typically appears as a band at approximately 33-34 kDa .

What are the optimal applications for WDR61 antibodies in research?

WDR61 antibodies have been validated for multiple applications in research settings. The most common applications include:

Optimization of dilutions for specific experimental conditions is recommended .

What is the recommended protocol for nuclear extraction when studying WDR61?

For nuclear extraction when studying WDR61, researchers can follow this validated protocol:

• Harvest cells by scraping in ice-cold PBS

• Extract at 4°C in buffer containing 50 mM Tris-HCl pH 7.5, 5 mM EDTA, 250 mM NaCl, and 0.1% NP-40 supplemented with protease and phosphatase inhibitors for 30 minutes

• Centrifuge at 13,000 rpm at 4°C for 1 hour

• Collect the supernatant and mix with two volumes of buffer containing 50 mM Tris-HCl pH 7.5, 5 mM EDTA, 100 mM NaCl, 0.1% NP-40, and 10% glycerol

This method has been successfully used in studies examining WDR61's nuclear interactions and functions .

How can WDR61 antibodies be used to investigate R-loop interactions and DNA damage?

Recent studies have revealed WDR61's critical interaction with R-loops and its potential role in genomic stability. To investigate these interactions, researchers can employ:

• Co-Immunoprecipitation (Co-IP): Use WDR61 antibodies to pull down protein complexes and analyze R-loop components using IP buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM MgCl₂, 0.5% NP-40, and 10% Glycerol) supplemented with protease and phosphatase inhibitors .

• Chromatin Immunoprecipitation (ChIP): Monitor WDR61 occupancy at specific genomic loci associated with R-loop formation.

• Immunofluorescence with DNA damage markers: Co-stain for WDR61 and DNA damage markers (γH2AX) to assess correlation between WDR61 loss and DNA damage accumulation following knockout or knockdown experiments .

• R-loop detection assays: Use the S9.6 antibody (recognizes RNA-DNA hybrids) in combination with WDR61 antibodies to examine co-localization or changes in R-loop accumulation upon WDR61 manipulation .

These approaches can help elucidate how WDR61 prevents R-loop accumulation and subsequent DNA damage in various cellular contexts.

What is the relationship between WDR61 and breast cancer, and how can antibodies help study this connection?

Researchers can use WDR61 antibodies to:

• Examine WDR61 expression levels in breast cancer tissues via IHC (validated at 1:50-1:500 dilution)

• Monitor WDR61 knockdown efficiency in functional studies

• Investigate downstream effects of WDR61 depletion on cellular signaling pathways

• Explore potential biomarker applications by correlating WDR61 expression with tumor characteristics and patient outcomes

Immunohistochemistry has successfully detected WDR61 in human breast cancer tissue, making this approach particularly valuable for translational research .

What are common issues in Western Blotting with WDR61 antibodies and how can they be resolved?

When performing Western blotting with WDR61 antibodies, researchers may encounter several challenges:

For optimal results, use chromatin fractionation protocols when focusing on nuclear WDR61, as this enriches for chromatin-associated proteins .

How should researchers prepare samples for optimal WDR61 detection in co-immunoprecipitation experiments?

For effective co-immunoprecipitation of WDR61 and its interacting partners, follow these methodological recommendations:

• Nuclear extract preparation:

  • Harvest cells in ice-cold PBS

  • Extract with buffer containing 50 mM Tris-HCl pH 7.5, 5 mM EDTA, 250 mM NaCl, and 0.1% NP-40 with protease/phosphatase inhibitors

  • Process as described in section 2.2

• Co-IP procedure:

  • Use 1.5-2 mg nuclear protein extract per reaction

  • Mix with 5 μg of WDR61 antibody and 50 μl of protein A magnetic Dynabeads

  • Perform in IP buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM MgCl₂, 0.5% NP-40, 10% Glycerol)

  • Wash beads three times with IP buffer and once with PBST

  • Elute proteins in 75 μl 2X SDS loading buffer with 50 nM DTT, heat at 95°C for 15 minutes

This protocol has been validated for studying WDR61's interactions with transcriptional machinery and R-loops .

How should researchers interpret changes in WDR61 expression in relation to R-loop formation and genomic stability?

When analyzing WDR61 expression changes and their relationship to R-loops and genomic stability, consider the following interpretive framework:

• Decreased WDR61 expression: Expected to correlate with increased R-loop accumulation, elevated DNA damage markers, and potentially reduced cell proliferation in cancer models .

• Increased WDR61 expression: May indicate enhanced R-loop resolution capacity and potentially contribute to genomic stability in normal cells, but could promote cancer cell survival in tumor contexts .

• Tissue-specific variations: WDR61's impact may vary across tissue types; validation in the specific biological system under study is crucial.

• Temporal dynamics: Consider whether observed effects are immediate responses to WDR61 modulation or adaptive responses developed over time.

When interpreting results, particularly in cancer studies, remember that WDR61 loss has been shown to compromise the proliferation of breast tumor cells with reduced colony-forming capacity, suggesting context-dependent functions .

What considerations should be made when analyzing WDR61's role in transcriptional regulation versus RNA surveillance?

WDR61 participates in two distinct but potentially overlapping cellular processes: transcriptional regulation (via PAF1C) and RNA surveillance (via the SKI complex) . When analyzing experimental data related to WDR61 function, researchers should consider:

• Functional separation: Design experiments that can distinguish between WDR61's roles in PAF1C versus SKI complex, potentially by examining specific binding partners unique to each complex.

• Context dependency: The predominant function of WDR61 may vary depending on cell type, developmental stage, or disease state.

• Compensatory mechanisms: Knockdown/knockout studies should account for potential compensatory upregulation of functionally related proteins.

• Downstream readouts: Measure appropriate endpoints for each pathway - transcriptional activity for PAF1C function and RNA decay rates for SKI complex function.

• Integrated analysis: Consider how disruption in one pathway (e.g., transcription) might affect the other (e.g., RNA surveillance) due to WDR61's dual roles.

The complex interplay between these functions likely contributes to the phenotypes observed when WDR61 is depleted, particularly in cancer contexts .