wtip Antibody

What is WTIP and why are antibodies against it important for research?

WTIP (Wilms Tumor 1 Interacting Protein) is a LIM domain scaffold protein that plays critical roles in cell adhesion dynamics and transcriptional responses. WTIP antibodies are essential research tools because:

• WTIP functions as a connector between cell adhesion changes and transcriptional responses, regulating cell phenotypic plasticity

• It localizes to focal adhesions in isolated cells and shifts to adherens junctions after cells make homotypic contacts

• WTIP has been implicated in embryonic development, podocyte function, and potentially tumor suppression

• Research indicates its downregulation is associated with poor prognosis in certain cancers like AML

Targeting different regions of WTIP with specific antibodies allows researchers to study its diverse cellular functions and protein-protein interactions in various physiological and pathological contexts.

What applications are WTIP antibodies suitable for in laboratory research?

WTIP antibodies can be utilized in multiple experimental applications depending on their specific characteristics:

When selecting a WTIP antibody for your specific application, consider the target epitope region, host species, clonality, and conjugation status to ensure optimal experimental outcomes .

How should I optimize immunofluorescence protocols for WTIP localization studies?

Optimizing immunofluorescence for WTIP localization requires careful attention to several parameters:

• Fixation method selection:

  • For cellular junctions and focal adhesions where WTIP localizes, 4% paraformaldehyde for 15 minutes at 4°C has been effective

  • Avoid overfixation which can mask epitopes and reduce signal intensity

• Permeabilization optimization:

  • For WTIP detection at cell junctions, use 0.2% Triton X-100 which improves antibody accessibility without disrupting structural integrity

  • Extended permeabilization (2 hours at 37°C) can enhance antibody penetration in dense tissues

• Antibody dilution and incubation:

  • Perform antibody titration experiments (typically 1:50 to 1:500 range) to determine optimal concentration

  • For primary WTIP antibodies, overnight incubation at 4°C may improve specific binding

  • Keep secondary antibody incubation to 1 hour at room temperature to minimize background

• Essential controls:

  • Include a no-primary antibody control to assess secondary antibody background

  • When possible, include WTIP knockdown samples as negative controls (e.g., using shWTIP as described in the literature)

  • Consider pre-immune serum controls when using polyclonal antibodies

Research demonstrates that WTIP localization changes depending on cell context—targeting to focal adhesions in isolated podocytes but shifting to adherens junctions after cell-cell contact formation . Ensuring proper sample preparation is critical for capturing these dynamic localization patterns.

What are the key considerations for optimizing WTIP antibody-based Western blotting?

For optimal Western blot results with WTIP antibodies, consider these methodological adjustments:

• Sample preparation:

  • Use RIPA buffer with protease inhibitors for efficient WTIP extraction

  • For complete denaturation, heat samples at 95°C for 5 minutes in reducing SDS-PAGE sample buffer

  • When studying WTIP interactions, consider using milder non-denaturing conditions

• Gel selection and transfer parameters:

  • WTIP is approximately 47 kDa, requiring 10-12% polyacrylamide gels for optimal resolution

  • Use wet transfer methods with 20% methanol for efficient protein transfer

• Blocking and antibody incubation:

  • 5% non-fat dry milk in TBST is generally effective for blocking

  • Optimize primary antibody concentration through titration experiments

  • Extend primary antibody incubation to overnight at 4°C for enhanced sensitivity

  • Keep secondary antibody incubation to 1 hour at room temperature

• Signal detection optimization:

  • Use enhanced chemiluminescence (ECL) for standard detection

  • For low abundance WTIP detection, consider using amplified ECL systems or fluorescent secondary antibodies with digital imaging

Studies have shown that WTIP expression can vary significantly between different cell types and disease states, with reduced expression observed in certain cancers like AML . Ensuring sufficient sensitivity in your detection method is crucial for accurate quantification.

What is the optimal immunoprecipitation protocol for studying WTIP protein interactions?

For effective immunoprecipitation of WTIP and its interaction partners, follow this optimized protocol:

• Antibody selection:

  • Choose antibodies with proven specificity for WTIP

  • Consider using antibodies targeting different WTIP regions to verify interactions

  • Both monoclonal and polyclonal antibodies can work, though polyclonals may capture more interaction partners

• Matrix choice and antibody immobilization:

  • For standard IPs, Protein A/G beads work well with rabbit polyclonal WTIP antibodies

  • Consider covalent crosslinking of antibody to beads if antibody co-elution interferes with detection of similarly sized interaction partners

  • Alternatively, use biotinylated WTIP antibodies with streptavidin beads for cleaner IPs

• Sample preparation and binding conditions:

  • Use mild lysis buffers (e.g., NP-40 or CHAPS-based) to preserve protein-protein interactions

  • Pre-clear lysates with beads alone to reduce non-specific binding

  • Optimize antibody-to-lysate ratio (typically 2-5 μg antibody per 500-1000 μg protein)

  • Allow sufficient binding time (2-4 hours at 4°C or overnight for weaker interactions)

• Washing and elution strategies:

  • Use multiple gentle washes with physiological buffers to remove non-specific proteins

  • For standard Western blot analysis, elute in SDS sample buffer

  • For maintaining complex integrity, consider milder elution with 0.1M glycine pH 2.5-3

Research has demonstrated successful co-immunoprecipitation of WTIP with binding partners like SSX2IP using this approach, with Flag-tagged WTIP constructs showing efficient complex formation .

How can I troubleshoot common issues with WTIP immunoprecipitation experiments?

When encountering problems with WTIP immunoprecipitation, consider these solution-oriented approaches:

Research studying WTIP interactions has shown that N-terminal fragments sometimes co-precipitate with binding partners more efficiently than full-length protein, suggesting domain-specific interactions that may require specialized approaches .

How can WTIP antibodies be used to study its role in podocyte biology and kidney disease?

WTIP antibodies serve as valuable tools for investigating podocyte function and renal pathophysiology:

• Localization studies in normal and disease states:

  • Immunofluorescence with WTIP antibodies reveals dynamic localization at podocyte cell junctions and focal adhesions

  • Immunogold electron microscopy using WTIP antibodies at 1:250 dilution can precisely localize WTIP within podocyte ultrastructure

  • Changes in WTIP distribution correlate with alterations in podocyte cytoskeletal architecture and cell-cell contacts

• Functional analyses:

  • Combining WTIP immunofluorescence with cytoskeletal markers helps correlate WTIP localization with actin dynamics and RhoA activity

  • Immunoprecipitation with WTIP antibodies can isolate protein complexes associated with slit diaphragm regulation

• Disease model applications:

  • WTIP antibodies can track protein redistribution in models of proteinuric kidney disease

  • Comparative studies between heterozygous and wild-type mice reveal WTIP's role in susceptibility to glomerular injury

  • Immunohistochemistry in human kidney samples shows correlation between WTIP expression patterns and disease progression

Research has demonstrated that WTIP knockout mice exhibit embryonic lethality, while heterozygous mice develop more significant proteinuria in response to injury models like LPS or adriamycin, highlighting WTIP's essential role in kidney development and function .

What approaches are used to investigate WTIP's potential tumor suppressor role using antibodies?

Recent research suggests WTIP may function as a tumor suppressor, particularly in hematological malignancies like AML . Antibody-based investigations include:

• Expression analysis in normal vs. malignant tissues:

  • Western blotting with WTIP antibodies shows reduced expression in AML cell lines and patient samples compared to controls

  • Quantitative analysis correlates WTIP expression levels with patient survival outcomes

• Mechanistic studies:

  • Immunoprecipitation with WTIP antibodies followed by mass spectrometry identifies novel interaction partners like FOXO3a

  • Chromatin immunoprecipitation (ChIP) using WTIP antibodies can reveal its involvement in transcriptional regulation

  • Co-immunofluorescence combining WTIP and FOXO3a antibodies demonstrates their co-localization patterns

• Functional validation approaches:

  • Comparing WTIP immunostaining in gain-of-function and loss-of-function models helps validate its tumor-suppressive effects

  • Antibody-based detection methods correlate WTIP levels with apoptotic markers like PUMA and caspase activation

Research has shown that WTIP expression is significantly downregulated in AML samples and associates with poor prognosis. Mechanistically, WTIP appears to upregulate FOXO3a and induces apoptosis through PUMA activation, suggesting potential therapeutic implications in targeting this pathway .

What controls should be included when using WTIP antibodies for experimental validation?

Proper controls are essential for generating reliable results with WTIP antibodies:

• Antibody specificity controls:

  • WTIP knockout/knockdown samples: Use tissues or cells with confirmed WTIP depletion (e.g., through CRISPR or shRNA)

  • Blocking peptide competition: Pre-incubate antibody with the immunizing peptide to confirm specificity

  • Multiple antibody validation: Use antibodies targeting different WTIP epitopes to confirm consistent findings

• Technical controls for immunodetection:

  • No primary antibody: Assess secondary antibody background

  • Isotype control: Use non-specific IgG of the same species and concentration

  • Pre-immune serum control: For polyclonal antibodies, use serum collected before immunization

  • Loading/transfer controls: Include housekeeping proteins for Western blots

• Positive controls:

  • Known WTIP-expressing tissues: Kidney podocytes show well-characterized WTIP expression

  • Overexpression systems: Cells transfected with WTIP expression constructs

• Biological context controls:

  • Developmental stages: WTIP expression appears during podocyte terminal differentiation

  • Treatment responses: WTIP localization changes with cell contact formation

Published research has employed shWTIP podocytes as negative controls, demonstrating that WTIP knockdown cells fail to spread normally and show altered focal adhesion patterns compared to control cells .

How can I optimize antibody concentration and incubation conditions for WTIP detection?

Optimizing antibody parameters is critical for balancing specific signal with minimal background:

• Antibody titration strategy:

  • Perform systematic dilution series (typically 1:50 to 1:2000) using consistent sample amounts

  • Evaluate signal-to-noise ratio rather than absolute signal intensity

  • For Western blots, aim for clean detection of the expected ~47 kDa WTIP band with minimal non-specific bands

  • For immunofluorescence, select dilutions that provide specific signal with minimal background fluorescence

• Incubation time and temperature optimization:

  • Primary antibodies:

    • Standard: 1-2 hours at room temperature

    • Enhanced sensitivity: Overnight at 4°C (especially for low abundance targets)

  • Secondary antibodies:

    • Usually 1 hour at room temperature is sufficient

    • Extended incubations can increase background without improving specific signal

• Buffer composition considerations:

  • Include 0.1-0.3% detergent (Triton X-100 or Tween-20) to reduce non-specific binding

  • Add 1-5% BSA or normal serum to block non-specific interactions

  • Consider using specialized blocking reagents for problematic samples

• Application-specific adaptations:

  • For paraffin sections, increased antibody concentration may be necessary after antigen retrieval

  • For thick tissue sections, extended incubation and permeabilization improve penetration

Published immunohistochemistry protocols for WTIP detection in kidney tissue have used rabbit polyclonal anti-WTIP antibodies at 1:50 dilution with satisfactory results .

How can multiplex immunostaining approaches be applied to study WTIP in complex tissues?

Multiplex immunostaining enables simultaneous visualization of WTIP alongside other markers, providing contextual information about its function:

• Technical considerations for WTIP multiplex staining:

  • Use primary antibodies from different host species to avoid cross-reactivity

  • Employ sequential staining protocols when using multiple antibodies from the same species

  • Carefully select fluorophores with minimal spectral overlap

  • Include single-stain controls to confirm antibody specificity and absence of bleed-through

• Validated multiplex combinations:

  • WTIP + synaptopodin: For podocyte-specific WTIP localization in kidney

  • WTIP + actin markers: To correlate WTIP with cytoskeletal dynamics

  • WTIP + FOXO3a + PUMA: To investigate tumor suppressor pathways

• Advanced multiplex approaches:

  • Tyramide signal amplification (TSA) for detecting low-abundance WTIP

  • Sequential immunofluorescence with antibody stripping for same-species antibodies

  • Spectral imaging and unmixing for resolving close fluorophores

Research has demonstrated that triple immunofluorescence combining standard methods for two antibodies with an antibody signal enhancer for WTIP produces improved immunolabeling results, particularly in challenging tissues .

What are the emerging applications of WTIP antibodies in studying developmental processes?

WTIP antibodies are increasingly used to investigate developmental biology:

• Embryonic development studies:

  • Research shows WTIP is essential for early murine embryonic development, with knockout mice exhibiting embryonic lethality

  • WTIP antibodies can track expression patterns in developing kidneys, heart, and eyes

  • Immunohistochemistry using β-galactosidase antibodies in heterozygous gene trap mice helps identify WTIP expression timing and localization

• Cell differentiation monitoring:

  • WTIP expression in podocytes initiates at the capillary loop stage, coinciding with terminal differentiation

  • Antibodies reveal WTIP's dynamic redistribution during morphogenetic processes

• Signaling pathway investigations:

  • Recent findings suggest WTIP interacts with the centrosomal maturation factor SSX2IP during neurulation

  • Co-immunoprecipitation and co-localization studies with antibodies against both proteins help elucidate these developmental mechanisms

• Disease model applications:

  • WTIP antibodies can assess how developmental defects contribute to congenital abnormalities

  • Comparative immunostaining between normal and pathological development provides insights into disease mechanisms