YES1 Antibody, FITC conjugated

What is the optimal fixation method for immunofluorescence detection using YES1-FITC antibody?

For optimal immunofluorescence results with YES1-FITC antibody, researchers should follow a standardized fixation protocol:

• Culture cells on coverslips or appropriate imaging surfaces to 70-80% confluence

• Fix cells with 4% paraformaldehyde in PBS for 15 minutes at room temperature

• Wash three times with PBS (5 minutes each)

• Permeabilize cells with 0.2% Triton X-100 in PBS for 5 minutes

• Wash twice with PBS (5 minutes each)

• Block with PBS containing 10% fetal bovine serum for 20 minutes

• Apply YES1-FITC antibody diluted 1:500 in blocking solution

• Incubate for 1 hour at room temperature in the dark

• Wash twice with PBS and mount for visualization using a fluorescence microscope with appropriate FITC filter

This approach preserves cellular architecture while allowing antibody access to the target protein.

Which cell types have been validated for YES1-FITC antibody applications?

YES1 antibodies have been successfully tested in multiple cell lines, allowing researchers to select appropriate experimental models:

These validations provide confidence for researchers planning experiments in these systems . When working with new cell types, preliminary validation experiments are recommended.

What is the recommended antibody dilution for different applications?

Optimal antibody dilutions vary by application method, and careful titration may be necessary for each experimental system:

These guidelines provide starting points, but researchers should optimize conditions for their specific experimental systems . For new applications or cell types, titration experiments are essential to determine the ideal antibody concentration.

How can YES1-FITC antibody be used to study protein-protein interactions in membrane complexes?

YES1-FITC antibody can be employed in sophisticated protein-protein interaction studies, particularly in membrane complexes:

• Co-localization studies: Using YES1-FITC antibody alongside antibodies against suspected interaction partners (such as CFTR or YAP1) labeled with spectrally distinct fluorophores allows for visualization of spatial relationships.

• Proximity Ligation Assay (PLA): This technique can detect protein interactions with YES1 at distances <40 nm by combining YES1-FITC antibody with non-FITC antibodies against interaction partners.

• FRET analysis: For interactions detected at molecular scale (1-10 nm), Förster Resonance Energy Transfer between FITC on YES1 antibody and acceptor fluorophores on antibodies targeting interaction partners provides quantitative measurement.

• Immunoprecipitation followed by fluorescence detection: YES1-FITC antibody can be used to precipitate protein complexes, with FITC signal serving as a confirmation of successful capture .

Research has demonstrated that YES1 forms complexes with F508del-CFTR at the plasma membrane, mediated by YAP1, with this interaction being specifically found in mutant but not wild-type CFTR protein complexes .

How can YES1-FITC antibody be applied in studies of YES1's role in disease mechanisms?

YES1-FITC antibody enables several advanced approaches for investigating YES1's role in pathological processes:

• Cystic fibrosis research: In studies of CFTR trafficking and function, YES1-FITC antibody can track YES1's association with corrector-rescued F508del-CFTR, revealing how YES1 mediates membrane removal of rescued channels. Flow cytometry or high-content imaging using the antibody can quantify this process in response to therapeutic interventions .

• Cancer research: YES1 overexpression has been observed in multiple cancer types. YES1-FITC antibody can be used in tissue microarray analysis to correlate YES1 expression patterns with clinical outcomes, particularly in lung and colon cancers where positive IHC detection has been validated .

• Infectious disease mechanisms: Since YES1 promotes Neisseria gonorrhoeae infectivity by phosphorylating MCP/CD46, YES1-FITC antibody can track YES1 recruitment to infection sites during pathogen-host interactions .

• Kinase inhibitor screening: Using YES1-FITC antibody in high-content screens allows visualization of YES1 localization changes in response to potential inhibitors like SU6656, facilitating development of therapeutic agents targeting YES1-mediated pathways .

What approaches can be used to validate YES1-FITC antibody specificity in experimental systems?

Rigorous validation of antibody specificity is critical for research integrity. For YES1-FITC antibody, several complementary approaches are recommended:

• Knockdown/knockout controls: Compare staining patterns in cells with normal YES1 expression versus those treated with validated YES1 siRNAs (achieving ~80% depletion). This approach confirms signal specificity and establishes background levels .

• Peptide competition assay: Pre-incubate YES1-FITC antibody with excess immunizing peptide (amino acids 450-480 of human YES1) before application to samples. Specific signal should be blocked while non-specific binding remains .

• Western blot correlation: Confirm that immunofluorescence patterns correlate with Western blot results showing the expected 61 kDa band in the same samples .

• Cross-species validation: Test reactivity across human, monkey, mouse, and rat samples to ensure consistent detection of the evolutionary conserved epitope .

• Technical controls: Include isotype control antibodies conjugated to FITC to distinguish specific from non-specific fluorescence signal.

What are the common causes of high background when using YES1-FITC antibody?

High background is a frequent challenge in immunofluorescence experiments. For YES1-FITC antibody, several specific factors may contribute:

• Insufficient blocking: Extend blocking time to 30-60 minutes with PBS containing 10% FBS, or test alternative blocking agents like 5% BSA or commercial blocking buffers .

• Suboptimal antibody dilution: Background often results from excessive antibody concentration. Perform a dilution series (1:250 to 1:2000) to determine optimal signal-to-noise ratio .

• Autofluorescence: Cellular components like NADH, flavins, and lipofuscin can emit in the FITC channel. Include unstained controls and consider autofluorescence quenching reagents if necessary.

• Fixation artifacts: Overfixation can increase non-specific binding. Optimize paraformaldehyde concentration (2-4%) and fixation time (10-20 minutes) .

• Storage conditions: FITC conjugates are sensitive to light and pH. Store antibody at -20°C and protect from light during all procedures to prevent degradation that can lead to non-specific binding .

• Sodium azide concentration: While 0.01% sodium azide is included as a preservative, higher concentrations may affect cell physiology or viability in live-cell applications .

How can researchers optimize dual immunostaining with YES1-FITC antibody and other markers?

Multi-color immunofluorescence requires careful planning to avoid signal overlap and optimize detection:

• Fluorophore selection: When combining with other antibodies, choose fluorophores with minimal spectral overlap with FITC (excitation ~495 nm, emission ~520 nm). Good companions include Cy3, Cy5, or Alexa Fluor 594/647.

• Sequential staining protocol:

  • Complete YES1-FITC antibody staining following standard protocol

  • Wash thoroughly (3-5 times) with PBS

  • Block again briefly (10 minutes)

  • Apply second primary antibody

  • Continue with detection using non-overlapping fluorophores

• Cross-reactivity testing: When studying YES1's interaction with other SRC family members, potential cross-reactivity must be evaluated. Pre-adsorb antibodies against recombinant proteins to ensure specificity .

• Imaging considerations: Acquire signals sequentially rather than simultaneously when using filter-based microscopy to prevent bleed-through. For confocal microscopy, adjust laser power and detector gain to minimize cross-channel contamination.

• Controls: Include single-stained samples for each antibody to establish proper compensation settings and confirm absence of cross-reactivity.

This approach has been successfully demonstrated in studies examining YES1 co-localization with YAP1 and CFTR at the plasma membrane .

What is the stability of YES1-FITC antibody under various storage and experimental conditions?

Understanding stability parameters ensures reliable experimental results:

FITC conjugates are particularly sensitive to pH changes and photobleaching. When planning extended imaging sessions, consider anti-fade mounting media to preserve signal intensity .

How should researchers quantify and normalize YES1-FITC signal in cellular compartments?

• Subcellular localization analysis:

  • Acquire z-stacks to capture the full cellular volume

  • Use cellular markers to define compartments (e.g., WGA for plasma membrane, DAPI for nucleus)

  • Apply automated segmentation to delineate compartments

  • Calculate intensity ratios between compartments (e.g., membrane/cytoplasm ratio)

• Normalization approaches:

  • Total cellular fluorescence: Normalize compartment-specific signal to total cellular fluorescence

  • Cell area: Account for size differences between cells

  • Reference protein: Co-stain with housekeeping protein markers for internal control

• Statistical considerations:

  • Analyze >30 cells per condition for robust statistics

  • Use appropriate statistical tests for distribution type (parametric vs. non-parametric)

  • Account for cell-to-cell variability with mixed models when appropriate

• Technical controls for quantification:

  • Include fluorescence standards to account for day-to-day variation

  • Apply flat-field correction to compensate for uneven illumination

  • Establish detection limits using YES1-depleted cells

How can researchers interpret changes in YES1 localization in response to experimental treatments?

Changes in YES1 localization often reflect functional alterations in signaling pathways:

• Plasma membrane to cytoplasm translocation: May indicate activation state changes or altered interaction with binding partners like YAP1. Quantify using membrane/cytoplasm intensity ratios measured along line profiles across cells .

• Nuclear translocation: YES1 can translocate to the nucleus in some contexts. Compare nuclear/cytoplasmic ratios and correlate with cell cycle markers or transcriptional changes.

• Punctate redistribution: Formation of distinct puncta may indicate YES1 recruitment to specific complexes or membrane domains. Analyze using particle counting algorithms to quantify number, size, and intensity of puncta.

• Temporal dynamics: For experiments tracking changes over time (e.g., after treatment with SU6656), analyze both magnitude and kinetics of redistribution to gain mechanistic insights .

• Correlation with functional outcomes: Correlate YES1 localization changes with functional measurements (e.g., CFTR-mediated ion transport) to establish physiological relevance of observed redistributions .

Research has demonstrated that inhibiting YES1 with SU6656 increases F508del-CFTR abundance at the cell surface, corresponding with enhanced CFTR-mediated ion transport, establishing a functional link between YES1 localization/activity and physiological outcomes .

How can YES1-FITC antibody be used in high-content screening applications?

YES1-FITC antibody can be adapted for high-content screening to identify modulators of YES1 function:

• Assay development workflow:

  • Optimize cell density in multi-well imaging plates

  • Establish automated immunostaining protocols compatible with liquid handling systems

  • Develop image analysis pipelines for quantifying YES1 parameters (intensity, localization, co-localization)

  • Validate with known controls (e.g., YES1 siRNA, SU6656 inhibitor)

• Parameters to measure:

  • Total YES1-FITC intensity per cell

  • Subcellular distribution patterns

  • Co-localization with interaction partners (e.g., YAP1, CFTR)

  • Downstream signaling effects (combining with phospho-specific antibodies)

• Application examples:

  • Screening corrector compounds for cystic fibrosis that prevent YES1-mediated removal of F508del-CFTR from the plasma membrane

  • Identifying novel YES1 kinase inhibitors with distinct effects compared to SU6656

  • Discovering compounds that disrupt YES1-YAP1 interaction

What considerations are important when using YES1-FITC antibody in live-cell imaging?

While most applications use fixed cells, live-cell applications require special considerations:

• Antibody delivery methods:

  • Microinjection: Direct delivery with precise control but technically challenging

  • Cell-penetrating peptide conjugation: Enhances membrane permeability

  • Electroporation: Temporary membrane permeabilization for antibody entry

  • Expression of intracellular antibody fragments (intrabodies): Genetic approach requiring additional validation

• Buffer considerations:

  • Use sodium azide-free formulations as azide inhibits cellular respiration

  • Optimize antibody concentration to minimize perturbation of cellular functions

  • Consider pH and osmolarity effects on live cells

• Phototoxicity management:

  • Minimize exposure times and light intensity

  • Use oxygen scavengers to reduce phototoxicity

  • Consider photobleaching effects in experimental design

• Alternative approaches to consider:

  • Fluorescent protein-tagged YES1 expression may provide better results for dynamic studies

  • SNAP-tag or HaloTag systems offer alternative labeling strategies with reduced impact on protein function

How can researchers apply YES1-FITC antibody in studying the Hippo signaling pathway through YES1-YAP1 interaction?

YES1-FITC antibody provides valuable tools for investigating the Hippo signaling pathway:

• Co-localization with YAP1:

  • Perform dual immunofluorescence with YES1-FITC and YAP1 antibodies

  • Quantify co-localization using Pearson's or Manders' coefficients

  • Analyze changes in co-localization following pathway stimulation or inhibition

• Functional studies:

  • Combine YES1-FITC staining with YAP1 nuclear localization analysis

  • Correlate YES1-YAP1 interaction with downstream transcriptional targets

  • Use in cells with manipulated Hippo pathway components to determine hierarchy

• Interaction dynamics:

  • Apply YES1-FITC antibody in FRAP (Fluorescence Recovery After Photobleaching) studies to assess dynamics of YES1-containing complexes

  • Combine with biosensors for Hippo pathway activity

  • Track changes following mechanical stress or cell density alterations that affect Hippo signaling

• PDZ-mediated interactions:

  • Investigate the tripartite complex of YES1-YAP1-NHERF1 using YES1-FITC antibody

  • Study how disruption of PDZ domains affects complex formation and localization

  • Correlate with CFTR regulation in relevant cell types

Research has demonstrated that YAP1 mediates the interaction between YES1 and F508del-CFTR, serving as an adaptor protein that associates with the SH3 domain of YES1 kinase and modulates its activity and plasma membrane localization through PDZ-mediated interaction with NHERF1 .