Phospho-FYN (Tyr530) Antibody

What is Phospho-FYN (Tyr530) Antibody and what specifically does it detect?

Phospho-FYN (Tyr530) Antibody is a polyclonal antibody specifically designed to detect endogenous levels of FYN protein only when phosphorylated at tyrosine 530. This antibody typically detects a protein with a molecular weight of approximately 59 kDa .

FYN is a non-receptor tyrosine-protein kinase that belongs to the Src family of cytoplasmic tyrosine kinases. It plays crucial roles in multiple biological processes, including:

• Regulation of cell growth and survival

• Cell adhesion and integrin-mediated signaling

• Cytoskeletal remodeling and cell motility

• Immune response mechanisms

• Axon guidance in neural development

The antibody is typically generated using a synthetic phosphopeptide derived from human FYN around the phosphorylation site of Tyrosine 530, often with the sequence motif P-Q-YP-Q-P .

What applications is Phospho-FYN (Tyr530) Antibody validated for?

Based on manufacturer specifications, Phospho-FYN (Tyr530) Antibody has been validated for multiple research applications:

The antibody shows cross-reactivity with human, mouse, and rat samples, making it versatile for comparative studies across these species .

How does phosphorylation at Tyr530 affect FYN activity and function?

Phosphorylation at Tyr530 plays a critical regulatory role in FYN kinase activity:

• Inactive state maintenance: Phosphorylation on the C-terminal tail at Tyr530 maintains the enzyme in an inactive state .

• Allosteric regulation: Evidence suggests Tyr530 serves as a de facto c-Src autophosphorylation site with slow time-resolution kinetics and a strong intermolecular component .

• Activity modulation: In experimental studies, a FYN Y530F mutant (preventing phosphorylation at this site) displays higher phospho-tyrosine activity and faster kinetics of other phosphorylation events compared to wild-type FYN .

• Signaling pathway involvement: The phosphorylation status at Tyr530 influences downstream pathways including T-cell receptor signaling, where protein tyrosine phosphatases like PTPRC/CD45 can dephosphorylate Tyr530, leading to activation .

• Structural effects: Phosphorylation creates an intramolecular interaction between the SH2 domain and the phosphorylated C-terminal tail, resulting in a closed, inactive conformation of the kinase .

What are the optimal storage conditions for Phospho-FYN (Tyr530) Antibody?

Proper storage is critical for maintaining antibody performance and specificity:

Additional recommendations:

• Aliquot upon receipt to minimize freeze-thaw cycles, which can degrade antibody performance

• Some formulations include 0.5% BSA to enhance stability and reduce non-specific binding

• When working with the antibody, keep it on ice and return to -20°C as soon as possible after use

• Monitor the expiration date, typically 12-24 months from date of receipt

How can researchers validate the specificity of Phospho-FYN (Tyr530) Antibody?

Validating antibody specificity is essential for reliable experimental results:

• Phosphatase treatment control: Treat samples with lambda phosphatase to remove phosphorylation, which should eliminate signal from a phospho-specific antibody.

• Phosphorylation site mutants: Use cells expressing FYN Y530F mutants as negative controls, which should show no reactivity with the phospho-specific antibody .

• Peptide competition assay: Pre-incubate the antibody with the phosphorylated peptide immunogen to block specific binding sites.

• Phosphatase inhibitor treatments: Compare samples treated with and without phosphatase inhibitors like Na₃VO₄:

  • Because Na₃VO₄ inhibits protein tyrosine phosphatases, it enhances Tyr530 phosphorylation

  • This results in increased signal with phospho-specific antibodies

• Cross-reactivity assessment: Test against other Src family members, as the antibody may cross-react with other Src family kinases due to conserved phosphorylation sites .

What are the recommended protocols for using Phospho-FYN (Tyr530) Antibody in cell-based assays?

For cell-based assays such as the Phospho-FYN (Tyr530) Fluorometric Cell-Based ELISA:

Sample Preparation Protocol:

• Cell selection and plating:

  • Choose cell lines that express detectable levels of FYN protein

  • For adherent cells, plate directly in 96-well plates

  • For suspension cells, coat plates with 100 μl of 10 μg/ml Poly-L-Lysine for 30 minutes at 37°C prior to cell seeding

  • Plate cells at 75-90% confluency for optimal results

• Fixation and permeabilization:

  • Use 4% formaldehyde (or 8% for suspension cells) to fix cells

  • Permeabilize with appropriate buffer to allow antibody access to intracellular targets

• Antibody incubation:

  • Add 50 μl of "Primary Antibody Mixture P" to corresponding wells for FYN (Phospho-Tyr530) detection

  • Add 50 μl of "Primary Antibody Mixture NP" to corresponding wells for total FYN detection

  • Cover and incubate for 16 hours (overnight) at 4°C, or 2 hours at room temperature for high-expression samples

• Detection and analysis:

  • Add secondary antibody mixture and incubate for 1.5 hours at room temperature

  • Wash thoroughly and read plates at appropriate wavelengths (Ex/Em: 651/667 for Dye 1 and 495/521 for Dye 2)

  • Normalize phospho-FYN signal to total FYN for accurate quantification of phosphorylation levels

How can Phospho-FYN (Tyr530) Antibody be used to study the allosteric regulation mechanisms in Src family kinases?

The study of allosteric regulation in Src family kinases requires sophisticated experimental approaches:

• Structural analysis integration: Combine antibody detection with structural biology approaches to correlate phosphorylation states with conformational changes:

  • Use Phospho-FYN (Tyr530) Antibody in conjunction with conformation-specific antibodies to detect inactive versus active states

  • Compare results with hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map allosteric networks

• Kinetic analysis protocols:

  • Implement time-course autophosphorylation assays to examine the kinetics of Tyr530 phosphorylation

  • As demonstrated in research, Tyr530 exhibits slow time-resolution kinetics and shows a strong intermolecular component in its phosphorylation pattern

• Mutagenesis approaches:

  • Generate tyrosine to phenylalanine mutants (Y530F) to study the impact on kinase activity

  • Experimental evidence shows Y530F mutants display higher phospho-tyrosine activity and faster kinetics of other phosphorylation events (e.g., Tyr419 and Tyr216) compared to wild-type FYN

• Multi-site phosphorylation analysis:

  • Use complementary phospho-specific antibodies targeting different sites (e.g., Tyr420 and Tyr530) to understand interdependence of phosphorylation events

  • Combine with mass spectrometry (LC-MS/MS) for comprehensive phosphorylation site mapping

What strategies can resolve conflicting results when detecting Phospho-FYN (Tyr530) across different sample types?

When facing discrepancies in Phospho-FYN (Tyr530) detection between different tissue types or experimental conditions:

• Sample preparation optimization:

  • Different tissue types may require specific lysis buffers to effectively extract phosphorylated proteins

  • For neural tissues: Use RIPA buffer with phosphatase inhibitor cocktails containing sodium fluoride, sodium orthovanadate, and β-glycerophosphate

  • For immune cells: Consider gentler NP-40 based lysis buffers with phosphatase inhibitors

• Isoform-specific considerations:

  • Account for tissue-specific alternative splicing of FYN

  • Neuronal FYN and hematopoietic FYN differ at the junction of the SH2 and kinase domains, which may affect epitope accessibility

• Normalization approaches:

  • Always normalize phospho-specific signals to total FYN protein levels

  • In cell-based ELISAs, include GAPDH detection as an internal positive control for normalizing target values

• Validation across techniques:

  • Confirm Western blot results with orthogonal methods such as immunoprecipitation followed by mass spectrometry

  • Consider using multiple antibody clones targeting the same phospho-epitope from different vendors

• Phosphatase activity assessment:

  • Different tissues exhibit varying levels of phosphatase activity that may affect Tyr530 phosphorylation

  • Implement graduated concentrations of phosphatase inhibitors to determine optimal conditions for each sample type

How does Phospho-FYN (Tyr530) detection contribute to understanding neurological disorders and immune dysfunction?

Phospho-FYN (Tyr530) research has significant implications for understanding disease mechanisms:

• Neurological disorders:

  • FYN is highly expressed in the brain (especially isoform 1) and plays critical roles in neural processes

  • Altered FYN phosphorylation states have been implicated in:

    • Alzheimer's disease pathogenesis through interactions with tau proteins

    • Synaptic plasticity disruption in models of neurodegeneration

    • Neuroinflammatory processes mediated by microglial activation

• Immune system dysregulation:

  • FYN isoform 2 is predominantly expressed in cells of hematopoietic lineages, especially T-lymphocytes

  • Phosphorylation at Tyr530 regulates:

    • T-cell receptor (TCR) signaling and activation

    • Negative feedback regulation through interactions with PAG1 and CSK recruitment

    • B-cell activation pathways critical for antibody production

• Experimental approaches:

  • Use phospho-specific antibodies in patient-derived samples to correlate disease states with FYN phosphorylation patterns

  • Implement genetic models (knock-in of phosphomimetic or phospho-deficient mutants) to recapitulate disease phenotypes

  • Correlate phosphorylation changes with functional outcomes using both in vitro and in vivo readouts

• Therapeutic implications:

  • Screen for compounds that modulate FYN phosphorylation status at Tyr530

  • Develop targeted approaches to normalize aberrant phosphorylation patterns in disease states

  • Monitor treatment efficacy using Phospho-FYN (Tyr530) Antibody as a biomarker for pathway engagement

What are common pitfalls when working with Phospho-FYN (Tyr530) Antibody and how can they be overcome?

Researchers frequently encounter these challenges when working with phospho-specific antibodies:

• High background in Western blots:

  • Optimize blocking conditions (try 5% BSA instead of milk, as milk contains phosphoproteins)

  • Increase washing time and volume

  • Titrate primary antibody concentration (typically 1:500-1:2000)

  • Use phospho-protein enrichment techniques prior to Western blot analysis

• Loss of phospho-signal during sample processing:

  • Add phosphatase inhibitors (e.g., Na₃VO₄, NaF, β-glycerophosphate) immediately during cell lysis

  • Keep samples cold throughout processing

  • Avoid repeated freeze-thaw cycles of samples

  • Consider using commercially available phosphoprotein preservation buffers

• Cross-reactivity with other Src family kinases:

  • The antibody may detect other Src family members when phosphorylated at homologous sites

  • Validate specificity using knockout or knockdown approaches

  • Perform immunoprecipitation with FYN-specific antibodies before probing with Phospho-FYN (Tyr530) Antibody

• Variable results between experiments:

  • Standardize cell culture conditions and lysis protocols

  • Ensure consistent activation/inhibition conditions when studying signaling pathways

  • Include positive controls (e.g., cells treated with known modulators of FYN phosphorylation)

  • Implement quantitative normalization to total FYN levels

How can researchers optimize detection sensitivity for low-abundance Phospho-FYN (Tyr530)?

Enhancing detection of low-abundance phosphorylated proteins requires specialized approaches:

• Sample enrichment strategies:

  • Immunoprecipitate total FYN before Western blotting with Phospho-FYN (Tyr530) Antibody

  • Use titanium dioxide (TiO₂) or immobilized metal affinity chromatography (IMAC) for phosphopeptide enrichment prior to analysis

  • Implement subcellular fractionation to concentrate FYN from relevant compartments (e.g., membrane fractions)

• Signal amplification methods:

  • For Western blotting: Use enhanced chemiluminescence (ECL) substrates designed for high sensitivity

  • For IHC/IF: Implement tyramide signal amplification (TSA) systems

  • For cell-based assays: Consider fluorometric ELISA approaches with high sensitivity dyes

• Instrument optimization:

  • Extend exposure times for Western blot imaging (while monitoring background levels)

  • Use high-sensitivity detection settings on automated imaging systems

  • Implement spectral unmixing for multiplexed fluorescence detection

• Pharmacological enhancement:

  • Treat samples with phosphatase inhibitors to preserve phosphorylation status

  • In some experimental contexts, use kinase activators to increase basal phosphorylation levels for better detection

What are the most effective multiplexing strategies when using Phospho-FYN (Tyr530) Antibody?

Multiplexed detection allows researchers to simultaneously analyze multiple parameters:

• Sequential immunoblotting approach:

  • First probe for Phospho-FYN (Tyr530)

  • Strip and reprobe for total FYN protein

  • Include additional markers (e.g., loading controls, other pathway components)

  • Use differently sized markers to avoid overlapping signals

• Multi-color fluorescence detection:

  • Use primary antibodies from different host species (e.g., rabbit anti-Phospho-FYN and mouse anti-total FYN)

  • Implement species-specific secondary antibodies conjugated to spectrally distinct fluorophores

  • Include nuclear counterstains or organelle markers for spatial context

• Cell-based ELISA multiplexing:

  • Utilize dual-wavelength detection systems:

    • Detect Phospho-FYN (Tyr530) with one fluorophore (e.g., Ex/Em: 651/667)

    • Simultaneously detect total FYN or GAPDH with another fluorophore (e.g., Ex/Em: 495/521)

  • This allows normalization and reduces well-to-well variation

• Mass cytometry (CyTOF) integration:

  • Label anti-Phospho-FYN (Tyr530) Antibody with rare earth metals

  • Combine with antibodies against other phosphorylated and total proteins

  • Enables high-dimensional analysis at single-cell resolution across diverse cell populations

• Multiplex IHC considerations:

  • Implement cyclic immunofluorescence or sequential immunohistochemistry

  • Use tyramide signal amplification with serial antibody stripping

  • Combine with tissue clearing techniques for 3D spatial analysis