WDR77 Monoclonal Antibody

What is WDR77 and what are its key functions in cellular processes?

WDR77, also known as MEP50 (Methylosome protein 50) or p44/Mep50, is a non-catalytic component of the 20S PRMT5-containing methyltransferase complex. It functions primarily in modifying specific arginines to dimethylarginines in several spliceosomal Sm proteins and histones . This modification targets Sm proteins to the survival of motor neurons (SMN) complex for assembly into small nuclear ribonucleoprotein core particles. WDR77 also plays important roles in transcriptional regulation and has been implicated in various cellular processes, including its recently discovered role in HBV replication restriction .

What epitopes of WDR77 are commonly targeted by commercial monoclonal antibodies?

Commercial WDR77 monoclonal antibodies typically target different regions of the full-length protein. Most commonly, antibodies are raised against recombinant human WDR77 protein fragments expressed in E. coli or against full-length recombinant WDR77 (AA 1-342 or AA 1-343) . When selecting an antibody for your research, consider the specific epitope recognition as it may affect detection efficiency in different applications or under various experimental conditions.

What species reactivity can be expected from WDR77 monoclonal antibodies?

Most commercial WDR77 monoclonal antibodies demonstrate cross-reactivity with human WDR77 . Some antibodies also show reactivity with mouse and rat WDR77, making them suitable for comparative studies across these species . When planning cross-species experiments, verify the specific reactivity profile of your selected antibody, as sequence homology variations between species may affect antibody performance.

What are the common applications for WDR77 monoclonal antibodies?

WDR77 monoclonal antibodies are applicable in various experimental techniques, including:

The specific application parameters may vary between different antibody products .

What are the optimal blocking conditions when using WDR77 monoclonal antibodies for Western blot analysis?

For Western blot applications using WDR77 monoclonal antibodies, optimal blocking typically involves 5% non-fat dry milk or 3-5% BSA in TBST (Tris-buffered saline with 0.1% Tween-20) for 1 hour at room temperature. This blocking step is critical to reduce background signal while maintaining specific detection of WDR77. For mouse-derived monoclonal antibodies, using mouse IgG-specific secondary antibodies can improve signal-to-noise ratio and prevent cross-reactivity with endogenous immunoglobulins in tissue samples.

How can I validate the specificity of WDR77 monoclonal antibodies in my experimental system?

Validating antibody specificity requires multiple approaches:

• Positive and negative controls: Use cell lines or tissues with known WDR77 expression levels.

• Knockdown/knockout validation: Compare antibody signal in WDR77 siRNA-treated cells versus control. The efficiency of WDR77 knockdown can be evaluated by RT-qPCR and Western blot analysis as demonstrated in previous studies .

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide to verify specific binding.

• Multiple antibody validation: Use antibodies from different clones or that target different epitopes.

• Recombinant protein detection: Test against purified recombinant WDR77 protein with known concentration.

How can WDR77 monoclonal antibodies be employed to study the PRMT5-WDR77 methyltransferase complex?

WDR77 monoclonal antibodies are valuable tools for investigating the PRMT5-WDR77 methyltransferase complex through:

• Co-immunoprecipitation (Co-IP): Use WDR77 antibodies to pull down the complex and identify associated proteins.

• Chromatin immunoprecipitation (ChIP): Determine the genomic binding sites of the complex, particularly in relation to H4R3me2s marks.

• Protein-protein interaction studies: Investigate how WDR77 interacts with PRMT5 and other proteins in the methyltransferase complex.

• Methyltransferase activity assays: Assess how depletion or overexpression of WDR77 affects PRMT5-mediated protein methylation.

Recent research has shown that WDR77 enhances PRMT5-triggered symmetric dimethylation of arginine 3 on H4 (H4R3me2s) on the cccDNA minichromosome to control cccDNA transcription in HBV studies .

What approaches can be used to investigate WDR77's role in HBV replication using monoclonal antibodies?

Based on recent discoveries about WDR77's role in HBV replication, researchers can:

• Infection models: Establish HBV de novo infection models in primary human hepatocytes (PHH) or HepG2-NTCP cells to study WDR77's function.

• Quantitative analysis: Use Western blot with WDR77 monoclonal antibodies to track protein levels during HBV infection.

• Chromatin studies: Apply ChIP assays using WDR77 antibodies to examine association with cccDNA.

• Protein degradation pathways: Investigate HBx-mediated degradation of WDR77 through the DDB1-containing E3 ubiquitin ligase pathway.

Research has demonstrated that HBV infection and HBx expression significantly reduce WDR77 protein levels in human liver-chimeric mice and HepG2-NTCP cells, while WDR77 knockdown elevates HBV DNA, HBsAg, HBeAg, and HBc levels in infection models .

How can I optimize chromatin immunoprecipitation protocols using WDR77 monoclonal antibodies?

For successful ChIP experiments with WDR77 monoclonal antibodies:

• Crosslinking optimization: Test different formaldehyde concentrations (0.75-1.5%) and incubation times (10-20 minutes).

• Sonication parameters: Adjust to achieve chromatin fragments of 200-500 bp.

• Antibody amount: Titrate WDR77 antibody (typically 2-5 μg per ChIP reaction).

• Beads selection: Protein A/G magnetic beads typically work well with mouse monoclonal antibodies.

• Washing stringency: Optimize salt concentrations in wash buffers to reduce background while maintaining specific signal.

• Elution conditions: Test different elution buffers for optimal chromatin recovery.

What are common causes of false-negative results when using WDR77 monoclonal antibodies in Western blotting?

Several factors can contribute to false-negative results:

When troubleshooting, include positive control samples with known WDR77 expression and verify protein loading with housekeeping protein detection.

How can I resolve high background issues when using WDR77 monoclonal antibodies in immunohistochemistry?

To reduce high background in IHC applications:

• Optimize blocking: Extend blocking time or increase blocking agent concentration.

• Antibody dilution: Test serial dilutions to determine optimal concentration.

• Endogenous peroxidase quenching: Ensure complete quenching with H₂O₂ treatment.

• Washing steps: Increase washing duration and frequency between steps.

• Secondary antibody specificity: Use species-specific secondary antibodies with minimal cross-reactivity.

• Tissue preparation: Optimize fixation time and antigen retrieval methods.

• Counterstain optimization: Adjust counterstain intensity to improve signal-to-noise ratio.

What strategies can address weak or inconsistent signals in WDR77 detection by immunofluorescence?

For improved immunofluorescence results:

• Fixation method: Compare paraformaldehyde, methanol, or acetone fixation.

• Permeabilization optimization: Test different detergents (Triton X-100, saponin) and concentrations.

• Antigen retrieval: Apply heat-induced or enzymatic antigen retrieval methods.

• Signal amplification: Consider tyramide signal amplification or biotin-streptavidin systems.

• Mounting media: Use anti-fade mounting media to preserve fluorescence.

• Image acquisition: Optimize exposure settings and use appropriate filter sets.

• Antibody concentration: Titrate primary antibody concentration (typically 1:100-1:400).

How is WDR77 involved in epigenetic regulation through the PRMT5 complex?

Current research indicates that WDR77 enhances PRMT5-mediated symmetric dimethylation of histone H4 at arginine 3 (H4R3me2s), an epigenetic mark associated with transcriptional repression . In the context of HBV infection, WDR77 enhances PRMT5-triggered H4R3me2s on the cccDNA minichromosome to control cccDNA transcription . This epigenetic regulatory mechanism suggests WDR77 might play broader roles in gene expression control through chromatin modification, making WDR77 monoclonal antibodies valuable tools for epigenetic research.

What emerging applications exist for studying WDR77 in disease mechanisms beyond HBV?

While recent research has established WDR77's role in HBV replication, its involvement in other disease mechanisms is being explored:

• Cancer biology: As an androgen receptor cofactor (p44), WDR77 may influence hormone-responsive cancers.

• Splicing dysregulation: WDR77's role in spliceosomal protein methylation suggests potential involvement in splicing-related disorders.

• Stem cell biology: The PRMT5-WDR77 complex may regulate pluripotency and differentiation programs.

• Neurodegenerative diseases: Potential involvement through SMN complex regulation.

Monoclonal antibodies targeting WDR77 will be instrumental in elucidating these disease connections.

How does post-translational modification affect WDR77 function and antibody recognition?

Post-translational modifications (PTMs) of WDR77 can alter its function and potentially affect antibody recognition. While comprehensive PTM mapping of WDR77 is still emerging, researchers should consider:

• Phosphorylation sites: May affect complex formation with PRMT5.

• Ubiquitination: HBx has been shown to trigger degradation of WDR77 through DDB1-containing E3 ubiquitin ligase .

• Methylation: Self-regulation within the methyltransferase complex.

• Epitope masking: PTMs may affect antibody binding efficiency.

When investigating these modifications, using antibodies targeting different epitopes can provide complementary information about WDR77 status.