wdr18 Antibody

What is WDR18 and why is it significant in cellular biology research?

WDR18 (WD repeat domain 18) is a 47 kDa protein containing six WD repeat domains that form tertiary propeller structures. It has significant roles in multiple cellular processes:

• Component of the Five Friends of Methylated CHTOP (5FMC) complex involved in transcriptional regulation

• Essential member of the PELP1 complex required for 28S rRNA maturation and pre-60S ribosomal subunit nucleoplasmic transit

• Collaborates with TopBP1 to facilitate DNA damage checkpoint signaling

• Promotes ATR-dependent Chk1 phosphorylation through direct interaction with TopBP1

WDR18's cellular localization is primarily nuclear (nucleolus and nucleoplasm), with lower levels detected in cytoplasmic and chromatin fractions . Its involvement in fundamental cellular processes including ribosome biogenesis, DNA damage responses, and transcriptional regulation makes it a valuable research target.

For optimal WDR18 detection in IHC applications:

• Antigen retrieval optimization:

  • Primary recommendation: TE buffer at pH 9.0

  • Alternative approach: Citrate buffer at pH 6.0

  • Heat-mediated retrieval is typically more effective than enzymatic methods for nuclear proteins like WDR18

• Antibody dilution:

  • Begin with manufacturer's recommended range (typically 1:50-1:500)

  • Perform titration experiments on positive control tissues (human lung tissue has been validated)

  • Optimize incubation time and temperature (typically overnight at 4°C or 1-2 hours at room temperature)

• Detection system selection:

  • For low abundance detection: Use amplification systems like tyramide signal amplification

  • For co-localization studies: Consider fluorescent secondary antibodies

  • For routine detection: HRP-polymer detection systems offer good sensitivity and low background

• Validated positive controls:

  • Human lung tissue

  • Human lung cancer tissue

  • Human ovary tumor tissue

Always include both positive and negative controls, and consider using tissues with known WDR18 expression patterns to validate staining specificity.

What are the key considerations for Western blot optimization when using WDR18 antibodies?

Successful Western blot detection of WDR18 requires careful optimization:

• Sample preparation:

  • Complete lysis is essential as WDR18 has multiple cellular localizations

  • For nuclear protein enrichment: Use specialized nuclear extraction buffers

  • Recommended positive control: Jurkat cell lysate has been validated

• SDS-PAGE conditions:

  • Use 10-12% gels for optimal separation near the 47 kDa range

  • Longer running times improve separation from common contaminants

  • Consider gradient gels for better resolution of WDR18 and potential binding partners

• Antibody optimization:

  • Starting dilution: 1:500-1:1000 in 5% BSA or milk solution

  • Incubation time: Overnight at 4°C typically yields better results than shorter room temperature incubations

  • Washing buffer: TBS-T (0.1% Tween-20) with at least 3-5 wash cycles

• Troubleshooting common issues:

  Problem	Potential Cause	Solution
  No signal	Insufficient protein	Increase loading amount (15-30 μg is typical for cell lysates)
  Multiple bands	Non-specific binding	Increase blocking time, try different blocking agents, increase antibody dilution
  Incorrect molecular weight	Post-translational modifications	Compare with positive control, confirm with immunoprecipitation

For specialized applications like studying WDR18 complexes, consider native-PAGE or formaldehyde crosslinking before lysis to preserve protein-protein interactions.

How can I validate the specificity of a WDR18 antibody for my experimental system?

Thorough antibody validation ensures reliable research results:

• Positive and negative controls:

  • Positive control: Jurkat cell lysates show consistent WDR18 expression

  • Knockdown validation: siRNA or shRNA-mediated depletion of WDR18 should reduce signal

  • Overexpression validation: WDR18 overexpression vectors should increase signal intensity

• Multiple detection methods:

  • Compare results across multiple techniques (WB, IHC, IF)

  • Use antibodies targeting different epitopes of WDR18

  • Consider mass spectrometry validation of immunoprecipitated proteins

• Species reactivity validation:

  • Confirmed reactivity: Human samples

  • Predicted reactivity: Mouse (75% sequence identity), Rat (74% sequence identity), Bovine

  • For non-validated species: Perform sequence alignment of the immunogen region

• Functional validation:

  • Immunodepletion of WDR18 should impair associated functions (e.g., TopBP1-mediated Chk1 phosphorylation)

  • Antibody should detect expected cellular localization patterns (primarily nucleoplasm with lower cytoplasmic levels)

If antibody specificity remains a concern, consider using tagged WDR18 constructs in parallel experiments as an alternative detection method.

What methods are recommended for studying the WDR18-TopBP1 interaction in DNA damage response pathways?

To effectively study the WDR18-TopBP1 interaction in DNA damage checkpoint signaling:

• Protein interaction analysis:

  • Co-immunoprecipitation: Use WDR18 antibodies to pull down the complex, then probe for TopBP1

  • GST pulldown assays: Research shows GST-WDR18 can pull down endogenous TopBP1

  • Note: Anti-TopBP1 antibodies may bind to the CAD domain and interfere with WDR18 interaction, making reciprocal IP challenging

• Domain mapping experiments:

  • Critical interaction region: WDR18 fragment 161-250 is necessary and sufficient for TopBP1 interaction

  • For dominant-negative approaches: Consider using the TopBP1-interacting domain of WDR18 to inhibit endogenous interactions

• Functional assays:

  • Chk1 phosphorylation assay: Monitor phosphorylation at S344 as readout of WDR18-TopBP1 functional interaction

  • AT70-induced phosphorylation: Useful trigger for studying this pathway in experimental systems

• Advanced imaging techniques:

  • FRET analysis: Tag WDR18 and TopBP1 with appropriate fluorophores to monitor interactions in living cells

  • PLA (Proximity Ligation Assay): Detect endogenous protein interactions with high specificity

The interaction is mediated through the C-terminal CAD domain of TopBP1, and experimental designs should account for this specific binding region .

How can I design experiments to investigate WDR18's role in ribosome biogenesis through the PELP1 complex?

To study WDR18's function in ribosome biogenesis:

• Complex composition analysis:

  • Immunoprecipitate WDR18 and analyze interacting partners via mass spectrometry

  • Confirm interactions with known PELP1 complex components using co-IP and Western blotting

  • Density gradient fractionation to isolate pre-ribosomal particles containing WDR18

• rRNA processing assays:

  • Pulse-chase labeling with 5-fluorouracil to track newly synthesized rRNA

  • Northern blot analysis for 28S rRNA maturation intermediates following WDR18 depletion

  • RT-qPCR for quantifying pre-rRNA processing efficiency

• Nucleolar-nucleoplasmic transport:

  • Fluorescence recovery after photobleaching (FRAP) to measure pre-60S ribosomal subunit mobility

  • Cellular fractionation to quantify 60S subunit distribution in nuclear versus cytoplasmic compartments

  • Immunofluorescence co-localization studies with nucleolar, nucleoplasmic, and pre-60S markers

• Functional domain analysis:

  • Create truncation mutants based on the six WD repeat domains

  • Rescue experiments in WDR18-depleted cells with domain mutants

  • Test binding capacity of mutants to PELP1 complex components

This experimental framework allows for comprehensive analysis of WDR18's specific contribution to ribosome maturation and nucleoplasmic transit .

What are the methodological considerations when using WDR18 antibodies for subcellular localization studies?

For accurate subcellular localization of WDR18:

• Fixation and permeabilization optimization:

  • For nucleolar detection: 4% paraformaldehyde fixation preserves nucleolar structure

  • For cytoplasmic detection: Gentle permeabilization with 0.1-0.2% Triton X-100

  • Consider methanol fixation as an alternative to unmask certain epitopes

• Co-localization markers selection:

  • Nucleolar markers: Fibrillarin, nucleolin

  • Nucleoplasmic markers: SC35, PML bodies

  • Cytoplasmic markers: α-tubulin

  • Dynein axonemal particle markers based on research context

• Advanced microscopy techniques:

  • Super-resolution microscopy (STED, STORM) for precise localization

  • Live-cell imaging with fluorescently tagged WDR18 to monitor dynamic localization

  • Z-stack acquisition and 3D reconstruction for complete spatial distribution

• Biochemical fractionation validation:

  • Complement imaging with subcellular fractionation

  • Analyze nuclear, nucleolar, chromatin, and cytoplasmic fractions by Western blot

  • Expect highest levels in nucleoplasm with lower detection in cytoplasmic and chromatin fractions

• Controls and validation:

  • Competing peptide controls to confirm antibody specificity

  • siRNA knockdown to validate signal reduction

  • Cell cycle synchronization as WDR18 localization may vary throughout cell cycle

These approaches provide complementary methods to establish the dynamic subcellular distribution of WDR18 under different cellular conditions.

How can I design experiments to study the functional relationships between WDR18's different protein complexes?

To investigate potential crosstalk between WDR18's roles in different protein complexes:

• Competitive binding analysis:

  • In vitro binding assays with purified components to test whether TopBP1 and PELP1 complex members compete for WDR18 binding

  • Structure-function analysis using the TopBP1-interacting domain (amino acids 161-250) to determine if this region overlaps with PELP1 complex binding

• Inducible system design:

  • Create cell lines with inducible DNA damage to trigger TopBP1-WDR18 interactions

  • Monitor effects on ribosome biogenesis during DNA damage response activation

  • Use nucleus-specific WDR18 tethering systems to sequester the protein from one complex to assess effects on other complexes

• Quantitative proteomics approaches:

  • SILAC or TMT labeling to quantify changes in WDR18 interactome after specific cellular stresses

  • Proximity labeling (BioID, APEX) with WDR18 as bait to identify context-dependent interactions

  • Cross-linking mass spectrometry to map protein interaction interfaces

• Functional readout assays:

  Complex	Functional Readout	Method
  TopBP1 complex	Chk1 phosphorylation	Western blot with phospho-specific antibodies
  PELP1 complex	28S rRNA maturation	Northern blot, RT-qPCR
  5FMC complex	ZNF148 desumoylation	SUMO-specific Western blots

• Domain-specific antibodies application:

  • Use antibodies targeting different regions of WDR18 to potentially discriminate between different complexes

  • C-terminal antibodies (aa 390-418) versus antibodies to the TopBP1-binding region (aa 161-250)

These approaches can help determine whether WDR18 functions independently in different complexes or serves as an integration point between DNA damage responses and ribosome biogenesis.

How do I troubleshoot weak or inconsistent signals when using WDR18 antibodies?

When facing detection challenges with WDR18 antibodies:

• Sample preparation optimization:

  • Ensure complete lysis by using stronger extraction buffers for nuclear proteins

  • Add protease inhibitors immediately before lysis

  • Maintain cold temperatures throughout sample processing

  • Consider phosphatase inhibitors as phosphorylation may affect antibody recognition

• Technical adjustments for Western blot:

  Issue	Adjustment	Rationale
  Weak signal	Reduce antibody dilution (1:250-1:500)	Increases antibody availability
  Weak signal	Extend primary antibody incubation (overnight at 4°C)	Allows more time for binding
  High background	Increase blocking time (2 hours to overnight)	Reduces non-specific binding
  Multiple bands	Try alternative blockers (5% BSA vs. milk)	Different blockers affect specificity

• Antibody selection considerations:

  • C-terminal targeting antibodies (aa 390-418) show good specificity

  • Consider antibodies validated for your specific application and species

  • For human samples, multiple antibodies have been validated

  • For rodent samples, verify cross-reactivity - mouse (75% sequence identity) and rat (74% identity)

• Detection system enhancement:

  • Super-sensitive ECL substrates for Western blot

  • Signal amplification systems for IHC/IF (tyramide, polymer systems)

  • Longer exposure times balanced against background development

Remember that WDR18 levels may vary by cell type, with Jurkat cells serving as a reliable positive control for human studies .

What are the critical considerations when designing WDR18 knockdown validation experiments?

For rigorous validation of WDR18 knockdown experiments:

• Knockdown strategy selection:

  • siRNA: For transient effects (3-7 days), useful for initial screening

  • shRNA: For stable knockdown, essential for long-term studies of ribosome biogenesis

  • CRISPR/Cas9: For complete knockout, but may be lethal due to WDR18's essential functions

• Validation methodology:

  • mRNA quantification: RT-qPCR with validated primers spanning exon-exon junctions

  • Protein depletion: Western blot with validated WDR18 antibodies (1:500-1:1000 dilution)

  • Functional readouts: Monitor known processes requiring WDR18 (Chk1 phosphorylation, 28S rRNA processing)

• Control design:

  • Non-targeting siRNA/shRNA controls

  • Rescue experiments with siRNA-resistant WDR18 expression constructs

  • Domain mutant rescue to map essential functional regions

• Phenotypic analysis:

  • Cell viability assessment (WDR18 depletion may affect cell survival)

  • Cell cycle analysis (potential G2/M accumulation due to checkpoint defects)

  • Ribosome profiles using sucrose gradients

For publications, at least two independent knockdown approaches should be used, with phenotypes validated by rescue experiments to confirm specificity.

What emerging techniques could advance the study of WDR18 function and interactions?

Cutting-edge approaches for WDR18 research:

• Structural biology approaches:

  • Cryo-EM analysis of WDR18-containing complexes (PELP1, 5FMC, TopBP1)

  • AlphaFold2 prediction and molecular dynamics simulations of WDR18 structure

  • Hydrogen-deuterium exchange mass spectrometry to map dynamic binding interfaces

• Genome-wide interaction studies:

  • CRISPR screens to identify synthetic lethal interactions with WDR18

  • BioID/APEX proximity labeling to map the complete WDR18 interactome

  • ChIP-seq analysis if WDR18 is involved in chromatin-associated functions

• Single-cell techniques:

  • Single-cell RNA-seq to identify cell state-dependent expression patterns

  • Live-cell single-molecule tracking to monitor WDR18 dynamics

  • Single-cell proteomics to quantify WDR18 complex stoichiometry

• Translational research approaches:

  • Patient-derived samples to investigate WDR18 in disease contexts

  • Development of small molecule inhibitors of WDR18-protein interactions

  • PROTAC degraders for selective WDR18 depletion

These emerging technologies could reveal new insights into WDR18's multifaceted roles in cellular homeostasis, DNA damage responses, and ribosome biogenesis.

What are the important considerations when designing experiments to study WDR18 post-translational modifications?

For comprehensive analysis of WDR18 post-translational modifications:

• PTM prediction and detection:

  • In silico prediction of potential modification sites (phosphorylation, SUMOylation, ubiquitination)

  • Phospho-specific antibodies if available, or general phospho-enrichment followed by Western blot

  • SUMO/Ubiquitin immunoprecipitation under denaturing conditions to detect modified WDR18

  • Mass spectrometry analysis after enrichment for specific modifications

• Functional analysis approaches:

  • Site-directed mutagenesis of predicted modification sites

  • Phosphomimetic mutations (S/T to D/E) and phospho-deficient mutations (S/T to A)

  • SUMOylation/ubiquitination site mutations (K to R)

  • Rescue experiments in WDR18-depleted backgrounds

• Condition-specific modification analysis:

  • DNA damage induction (UV, hydroxyurea, IR)

  • Cell cycle synchronization to identify cell cycle-dependent modifications

  • Ribosomal stress induction (actinomycin D at low doses)

  • Proteasome inhibition to detect unstable modified forms

• PTM crosstalk investigation:

  • Sequential immunoprecipitations to detect proteins with multiple modifications

  • Inhibitor studies (kinase inhibitors, SUMO inhibitors) to determine modification hierarchies

  • Modification-specific interactome analysis using modified versus unmodified protein as bait