WDR73 Antibody, FITC conjugated

What is WDR73 and why is it a significant research target?

WDR73 is a WD40-repeat domain protein that plays crucial roles in microtubule functions and spindle poles during mitotic cell division. Its significance stems from its association with Galloway-Mowat syndrome (GAMOS), a rare genetic disorder characterized by the co-occurrence of neurological symptoms (including microcephaly, cerebral atrophy, and neural migration defects) and glomerular-renal disease. WDR73 mutations have been identified as causative factors in this syndrome, making it an important target for understanding disease mechanisms in both neurological and renal contexts .

What are the primary applications for WDR73 Antibody, FITC conjugated?

The FITC-conjugated WDR73 antibody is primarily utilized for:

• Flow cytometry (FACS) for analyzing WDR73 expression in cell populations

• Immunofluorescence (IF) for visualizing subcellular localization

• ELISA assays for quantitative protein detection

• Fluorescence-based Western blotting

This conjugated antibody eliminates the need for secondary antibody incubation in fluorescence-based applications, particularly beneficial for multicolor immunofluorescence studies .

What is the typical reactivity and specificity of commercially available WDR73 antibodies?

Most commercially available WDR73 antibodies, including FITC-conjugated versions, demonstrate specificity for human WDR73. According to product specifications, these antibodies typically recognize epitopes in the N-terminal region, particularly amino acids 44-73, and are validated through Western blotting against human samples. Specificity is typically confirmed through overexpression studies and shRNA knockdown validation .

How should researchers optimize fixation protocols when using WDR73 Antibody, FITC conjugated for immunofluorescence?

For optimal immunofluorescence results with FITC-conjugated WDR73 antibody:

• Fixation method selection: Both paraformaldehyde (PFA) and methanol fixation protocols have been successfully utilized, but their effectiveness varies depending on cell type and subcellular localization interests.

  • For cytoskeletal association studies: 4% paraformaldehyde (20 minutes at room temperature) preserves cytoskeletal structures better

  • For nuclear/chromatin studies: Ice-cold 100% methanol (5 minutes) provides superior nuclear antigen accessibility

• Permeabilization optimization: After PFA fixation, use 0.5% Triton X-100 in PBS for 15 minutes to ensure adequate antibody penetration, particularly for nuclear antigens

• Blocking parameters: 5% bovine serum albumin in PBS for 1 hour at room temperature effectively minimizes non-specific binding

• WDR73 antibody dilution range: Optimal dilutions typically fall between 1:50-1:200 for immunofluorescence applications .

What controls should be included when validating WDR73 Antibody, FITC conjugated specificity?

A robust validation protocol requires multiple controls:

• Negative controls:

  • WDR73 knockout/knockdown cells (CRISPR/Cas9 or shRNA approaches)

  • Isotype control antibodies (rabbit IgG-FITC conjugated)

  • Primary antibody omission control

• Positive controls:

  • Cells overexpressing recombinant WDR73 (documented in validation studies)

  • Tissues with known WDR73 expression (cerebral cortex, hippocampus, kidney glomeruli)

• Specificity validation:

  • Peptide competition assays using the immunizing peptide (AA 44-73)

  • Comparison with non-conjugated WDR73 antibody followed by FITC-conjugated secondary antibody

  • Dual staining with alternative WDR73 antibodies recognizing different epitopes .

What are the recommended protocols for using WDR73 Antibody, FITC conjugated in flow cytometry?

For optimal flow cytometry results:

• Sample preparation:

  • For cell suspensions: 1×10⁶ cells per sample

  • Fixation: 4% paraformaldehyde (10-15 minutes, room temperature)

  • Permeabilization: If needed for intracellular staining, use 0.1% saponin or commercial permeabilization buffer

• Antibody incubation:

  • Dilution range: 1:10-1:50 (higher concentration than for IF)

  • Incubation time: 30-60 minutes at 4°C in the dark

  • Washing: 3× with PBS containing 1% FBS

• Controls and gating strategy:

  • Unstained cells for autofluorescence baseline

  • Isotype-FITC control for non-specific binding

  • Begin gating on leukocytes when working with blood samples

  • Additional surface markers may be required to identify specific cell populations

• Storage precautions: Do not freeze FITC-conjugated antibodies; store at 4°C protected from light .

How can WDR73 Antibody, FITC conjugated be utilized to investigate Integrator complex interactions?

WDR73 has been shown to interact with INTS9 and INTS11 components of the Integrator complex. To investigate these interactions:

• Co-localization studies:

  • Use multi-color immunofluorescence with FITC-conjugated WDR73 antibody and differently conjugated antibodies against INTS9, INTS11, or other Integrator components

  • Analyze nuclear co-localization using confocal microscopy with Z-stack acquisition

  • Quantify co-localization using Pearson's or Mander's correlation coefficients

• Cell synchronization strategies:

  • Since WDR73-Integrator interactions may be cell cycle-dependent, synchronize cells using double-thymidine block protocols

  • Analyze different cell cycle phases separately

• Proximity ligation assay (PLA) adaptation:

  • Combine FITC-WDR73 antibody with antibodies against Integrator components

  • Use PLA probes compatible with FITC fluorescence to visualize protein-protein interactions in situ .

What approaches can be used to investigate WDR73's role in microtubule regulation using the FITC-conjugated antibody?

To study WDR73's microtubule regulatory functions:

• Live-cell imaging:

  • Microinjection of FITC-conjugated WDR73 antibody into live cells

  • Time-lapse microscopy during mitosis to track spindle pole localization

• Co-localization with tubulin:

  • Dual immunofluorescence with FITC-WDR73 antibody and α/β/γ-tubulin antibodies

  • Quantitative analysis of co-localization at mitotic structures

• Functional perturbation studies:

  • Combine with tubulin-targeting drugs (nocodazole, taxol) to disrupt microtubule dynamics

  • Analyze changes in WDR73 localization patterns

  • Correlate with cell cycle progression abnormalities

• WDR73 mutant expression effects:

  • Compare wild-type and GAMOS-associated mutant WDR73 localization patterns

  • Quantify effects on spindle formation and cell division .

How can WDR73 Antibody, FITC conjugated be applied to study focal adhesion in podocytes?

Recent research has implicated WDR73 in focal adhesion regulation, particularly in podocytes. To investigate this:

• Podocyte-specific staining protocols:

  • For immortalized podocyte cell lines: Use FITC-WDR73 antibody (1:50-1:100) with phalloidin counterstaining to visualize actin filaments

  • For kidney tissue sections: Combine with podocyte markers (synaptopodin, nephrin) to identify podocytes specifically

• PIP4K2C-WDR73 interaction analysis:

  • Dual staining with FITC-WDR73 antibody and PIP4K2C antibodies

  • Analyze effects of WDR73 depletion on PIP4K2C expression and stability

  • Quantify PIP2 levels in relation to WDR73 expression

• Focal adhesion quantification:

  • Measure number, size, and distribution of focal adhesions in control versus WDR73-depleted podocytes

  • Correlate with changes in cell morphology, particularly process formation and retraction .

What are common issues encountered with FITC-conjugated antibodies and their solutions?

Several technical challenges may arise:

• Photobleaching:

  • Problem: FITC is relatively susceptible to photobleaching during prolonged imaging

  • Solution: Use anti-fade mounting media containing DABCO or propyl gallate; minimize exposure during image acquisition; consider using alternative more photostable fluorophores for long-term imaging studies

• Background fluorescence:

  • Problem: High autofluorescence in certain tissues (especially kidney) in the FITC channel

  • Solution: Implement additional blocking steps with normal serum (5-10%); use Sudan Black B (0.1-0.3%) post-staining to reduce autofluorescence; consider spectral unmixing during image acquisition

• pH sensitivity:

  • Problem: FITC fluorescence is pH-dependent and decreases in acidic environments

  • Solution: Maintain buffers at pH 7.2-8.0; avoid acidic fixatives; use pH-stable mountants

• Signal amplification challenges:

  • Problem: Direct conjugates may have lower sensitivity than indirect detection

  • Solution: For weak signals, consider tyramide signal amplification (TSA) compatible with FITC fluorescence; ensure adequate permeabilization for intracellular targets .

How should conflicting localization data between different WDR73 antibodies be reconciled?

When facing discrepancies in WDR73 localization patterns:

• Epitope mapping analysis:

  • Different antibodies may recognize distinct epitopes, potentially masked in certain conformations or protein complexes

  • Compare antibodies targeting N-terminal versus C-terminal regions

• Validation hierarchy:

  • Prioritize localization data obtained from antibodies validated by multiple methods (knockout controls, overexpression, peptide competition)

  • Consider complementary approaches (GFP-tagged WDR73 expression) to confirm localization

• Cell-type and context dependency:

  • WDR73 localization may genuinely differ between cell types and physiological states

  • FITC-conjugated WDR73 antibody has shown both nuclear and cytoplasmic distribution, which aligns with its multiple reported functions

  • Standardize experimental conditions including cell cycle stage, confluency, and fixation protocols .

How can researchers distinguish between specific and non-specific binding in WDR73-FITC antibody applications?

To ensure detection specificity:

• Signal pattern analysis:

  • Specific WDR73 staining should show:

    • Enrichment at mitotic spindles during cell division

    • Nuclear and/or cytoplasmic localization patterns consistent with published data

    • Co-localization with known interaction partners (tubulin, INTS9/11)

  • Non-specific patterns often appear as:

    • Uniform cytoplasmic staining without subcellular enrichment

    • Excessive membrane or nucleolar signals

    • Persistence in knockout/knockdown controls

• Quantitative validation approaches:

  • Establish signal-to-noise ratios across multiple experiments

  • Implement intensity thresholding based on negative controls

  • Perform peptide competition assays at increasing peptide concentrations

• Multi-method confirmation:

  • Verify key findings with orthogonal techniques (Western blotting, mass spectrometry)

  • Use alternative antibodies targeting different WDR73 epitopes

  • Compare results from direct FITC conjugates with traditional primary-secondary antibody detection .

How is WDR73 Antibody, FITC conjugated being applied in studies of Galloway-Mowat syndrome pathogenesis?

Current research applications include:

• Patient-derived cell analysis:

  • FITC-WDR73 antibody enables visualization of mutant protein localization and abundance in patient fibroblasts and induced pluripotent stem cell (iPSC)-derived podocytes and neurons

  • Flow cytometric quantification of expression levels across different patient mutations

• Animal model validation:

  • Immunofluorescence analysis of conditional Wdr73 knockout mouse tissues

  • Correlation of WDR73 expression patterns with podocyte foot process injury and albuminuria development

• Mechanistic pathway investigations:

  • Visualization of WDR73's interaction with the autophagy-lysosomal pathway

  • Analysis of cell cycle progression defects in patient cells

  • Characterization of focal adhesion abnormalities in podocytes carrying WDR73 mutations .

What emerging techniques are being developed that incorporate WDR73 Antibody, FITC conjugated?

Innovative methodological approaches include:

• Super-resolution microscopy applications:

  • STED and STORM microscopy for nanoscale localization of WDR73 at microtubule structures

  • Single-molecule tracking of WDR73 dynamics during cell division

• Multi-omics integration strategies:

  • Fluorescence-activated cell sorting (FACS) with FITC-WDR73 antibody followed by transcriptomics/proteomics

  • Combining with proximity labeling approaches (BioID, APEX) to identify context-specific interaction partners

• Functional screening platforms:

  • High-content screening of drug libraries using automated imaging of WDR73-FITC staining patterns

  • CRISPR-based genetic modifier screens coupled with WDR73 immunofluorescence phenotyping .

How is WDR73 research contributing to understanding broader mechanisms of cell biology?

WDR73 research is illuminating:

• Integrator complex regulation:

  • WDR73's interaction with INTS9/11 suggests it functions in UsnRNA processing

  • FITC-WDR73 antibody enables visualization of dynamic interactions with Integrator components during transcriptional responses

• Post-mitotic cell maintenance mechanisms:

  • Both podocytes and neurons are terminally differentiated cells affected by WDR73 mutations

  • WDR73 studies reveal common pathways critical for maintaining these specialized cell types

• Microtubule-associated protein networks:

  • WDR73 interacts with α-, β-, and γ-tubulin, HSP-70, HSP-90, and other proteins

  • FITC-conjugated antibody allows visualization of these interaction networks in relevant cellular contexts

• Phosphoinositide signaling in cell adhesion:

  • Recent research indicates WDR73 regulates PIP4K2C stability and subsequent PIP2 production

  • This pathway appears critical for focal adhesion formation in podocytes, potentially explaining the kidney manifestations in GAMOS .