WDPCP Antibody, FITC conjugated

What is WDPCP and why is it significant in cellular research?

WDPCP (WD repeat-containing planar cell polarity effector) is a protein that plays dual critical roles in both planar cell polarity (PCP) and ciliogenesis. Its significance stems from its ability to regulate cell polarity and alignment through direct modulation of the actin cytoskeleton . WDPCP-deficient models exhibit phenotypes resembling Bardet-Biedl/Meckel-Gruber ciliopathy syndromes, including cardiac outflow tract and cochlea defects associated with PCP perturbation . The protein serves as a crucial link between cytoskeletal organization and cellular polarity, making it an important target for developmental biology and cell migration research.

Where is WDPCP protein localized in cells?

WDPCP demonstrates two primary localizations within cells:

In wild-type cells, WDPCP shows extensive colocalization with actin filament bundles (stress fibers) as visualized by phalloidin staining . Additionally, WDPCP is enriched in the cell cortex, where it colocalizes with vinculin at points of actin filament insertion into focal adhesions .

What is the optimal immunostaining protocol for WDPCP-FITC antibody in ciliogenesis studies?

For optimal detection of WDPCP in ciliogenesis studies, the following protocol modifications should be implemented:

• Cell preparation: Serum starve MEFs or relevant cell types for 24-48 hours to induce cilia formation

• Fixation: Use paraformaldehyde (4%) to maintain cellular architecture

• Primary antibody considerations:

  • For dual staining with ciliary markers: combine WDPCP-FITC antibody with antibodies against acetylated α-tubulin (1:1,000) and γ-tubulin (1:1,000)

  • For transition zone studies: combine with antibodies against Sept2 (1:1,000), Mks1 (1:2,000), Ift88 or Nphp1

• Visualization approach: For FITC-conjugated antibodies, use appropriate filter sets (excitation ~495nm, emission ~519nm)

• Controls: Include WDPCP-deficient cells (such as the Wdpcp Cys40 mutant) to confirm antibody specificity

How can I quantitatively assess WDPCP localization in actin cytoskeleton studies?

To quantitatively analyze WDPCP association with the actin cytoskeleton:

• Triple staining approach:

  • Phalloidin for actin visualization

  • WDPCP-FITC antibody for protein detection

  • Anti-Sept2 for colocalization analysis

• Image acquisition parameters:

  • Use high-resolution confocal microscopy (minimum resolution: 150-200nm)

  • Capture Z-stacks to fully evaluate cytoskeletal associations

  • Apply consistent exposure settings across experimental groups

• Quantification methods:

  • Measure phalloidin staining intensity (reduction of ~28% is observed in WDPCP-deficient cells)

  • Evaluate stress fiber morphology and thickness

  • Assess Sept2 distribution patterns (beaded "o" and "c" configurations indicate disrupted actin association)

  • Analyze focal adhesion morphometrics (size, shape, intensity)

How does WDPCP interact with Sept2, and what experimental approaches can verify this interaction?

WDPCP and Sept2 interaction can be verified through multiple complementary approaches:

• Co-immunoprecipitation experiments:

  • Express FLAG-tagged WDPCP (either transiently or stably) in an appropriate cell line

  • Transfect cells with Sept2-GFP construct

  • Perform immunoprecipitation using anti-FLAG antibody

  • Analyze precipitates by Western blotting with anti-GFP and anti-Sept2 antibodies

• Direct visualization of colocalization:

  • Perform triple immunostaining with anti-WDPCP (FITC-conjugated), anti-Sept2, and phalloidin

  • Use confocal microscopy to identify regions of colocalization in actin stress fibers

Research findings demonstrate that both Sept2-GFP and endogenous Sept2 are incorporated into FLAG-WDPCP-containing protein complexes, confirming their interaction .

What experimental approaches can determine the functional consequences of WDPCP deficiency on actin cytoskeleton?

To assess WDPCP's role in actin cytoskeleton regulation:

• Stress fiber analysis:

  • Compare wild-type and WDPCP-deficient cells stained with phalloidin

  • Quantify stress fiber thickness, alignment, and density

  • Assess Sept2 localization patterns in relation to actin filaments

• Focal adhesion characterization:

  Parameter	Wild-type	WDPCP-deficient	Statistical Significance
  Average size	0.37 μm²	0.32 μm²	p<0.001
  Mean vinculin intensity	100.2	157.8	p<0.01
  Length	1.18 μm	0.76 μm	p<0.0001
  Roundness	37.7	58.9	p<0.0001

• Cell migration assays:

  • Wound healing/scratch assays to assess directional migration

  • Time-lapse imaging to monitor membrane ruffling (reduced in WDPCP mutants)

  • Analysis of cell polarization during directed migration

What are the optimal storage and handling conditions for FITC-conjugated antibodies?

For maximum stability and performance of FITC-conjugated antibodies:

• Storage conditions:

  • Store at -20°C in aliquots to minimize freeze-thaw cycles

  • Stable for one year after shipment when properly stored

  • Aliquoting is unnecessary for -20°C storage of small quantities (20μl)

• Buffer composition:

  • Typically stored in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

  • Small size preparations (20μl) may contain 0.1% BSA for additional stability

• Light exposure considerations:

  • Minimize exposure to light during handling to prevent photobleaching

  • Store in amber or foil-wrapped tubes

  • During experimental procedures, protect from direct light when not imaging

What dilution ranges are recommended for FITC-conjugated antibodies in different applications?

Optimal dilution ranges vary by application:

These recommendations should be used as starting points, and researchers should optimize dilutions for their specific experimental systems to obtain optimal results .

How can I distinguish between specific WDPCP staining and background when using FITC-conjugated antibodies?

To maximize signal-to-noise ratio and confirm specificity:

• Essential controls:

  • WDPCP-deficient cells (such as Wdpcp Cys40 mutant) to establish baseline

  • Secondary-only controls to assess autofluorescence

  • Competitive blocking with immunizing peptide

  • Wild-type positive controls with known WDPCP expression patterns

• Signal verification approaches:

  • Confirm expected cellular localization (transition zone, actin stress fibers)

  • Verify colocalization with known interaction partners (Sept2)

  • Assess cellular phenotypes consistent with published WDPCP functions

  • Compare staining patterns with published immunohistochemistry data

• Background reduction strategies:

  • Optimize blocking conditions (5% BSA or 10% serum from species unrelated to primary/secondary antibodies)

  • Increase washing duration/volume

  • Titrate antibody concentration to minimize non-specific binding

  • Consider autofluorescence quenching methods if tissue-specific autofluorescence is problematic

What methodology should be used to investigate WDPCP's role in planar cell polarity in tissue sections?

For investigation of WDPCP's role in planar cell polarity:

• Tissue preparation:

  • For cochlear analysis: Dissect cochleae and stain with phalloidin and antibodies against WDPCP and Vangl2

  • For neural tube analysis: Perform cryo-sectioning and immunostain with WDPCP antibody alongside markers such as FoxA2 (1:1,000), Pax6 (1:1,000), and Olig2 (1:1,000)

• Analysis approach:

  • Examine kinocilia orientation in cochlear sections

  • Assess cell alignment patterns in epithelial tissues

  • Quantify protein distribution asymmetry across cell membranes

  • Compare wild-type patterns with WDPCP-deficient tissues

• Interpretation framework:

  • PCP defects in WDPCP mutants appear to result from direct disruption of the actin cytoskeleton rather than loss of cilia

  • WDPCP mutant cochlea exhibits normal kinocilia but shows PCP defects

  • Changes in focal adhesion morphology and actin organization contribute to cell migration and polarity defects

How can FITC-conjugated WDPCP antibodies be used to investigate novel protein interactions?

Strategies for identifying new WDPCP interaction partners:

• Proximity labeling approaches:

  • Combine WDPCP-FITC antibody localization data with BioID or APEX2 proximity labeling

  • Correlate spatial distribution with other cytoskeletal regulatory proteins

  • Verify potential interactions with co-immunoprecipitation following the established protocols for Sept2

• Live-cell dynamics analysis:

  • Use FITC-WDPCP antibody in conjunction with other fluorescently-tagged proteins

  • Track temporal recruitment patterns during cell migration or ciliogenesis

  • Correlate WDPCP redistribution with actin remodeling events

• Super-resolution microscopy applications:

  • Employ techniques like STORM or PALM to resolve nanoscale protein associations

  • Map WDPCP distribution relative to focal adhesion components and septin structures

  • Analyze structural changes in actin organization at higher resolution than conventional confocal microscopy

What methodological approaches can enhance detection sensitivity when working with low WDPCP expression levels?

For enhanced detection of low-abundance WDPCP:

• Signal amplification methods:

  • Tyramide signal amplification compatible with FITC fluorophores

  • Multiple antibody layering techniques

  • Consider stable expression systems similar to those used in the FLAG-WDPCP construction for experimental controls

• Sample preparation optimization:

  • Transiently transfected cells typically show very low protein abundance compared to stably transfected cells

  • Use concentrated protein samples for biochemical analyses

  • Consider cell synchronization to capture peak expression periods

• Detection system enhancements:

  • High-sensitivity cameras with increased quantum efficiency

  • Confocal systems with spectral unmixing capabilities

  • Deconvolution algorithms to improve signal-to-noise ratio

These methodological approaches can significantly improve detection of WDPCP in challenging experimental systems, enabling more detailed analysis of its molecular interactions and cellular functions.