P2RY1 Antibody, FITC conjugated

What is P2RY1 and why is it significant in research?

P2RY1 (purinergic receptor P2Y1) is a 373-amino acid G-protein coupled receptor with a molecular weight of approximately 42 kDa. It functions as a receptor for extracellular adenine nucleotides, particularly ADP. The receptor is membrane-associated and undergoes post-translational modifications, including glycosylation . P2RY1 plays a critical role in platelet activation through Gq-coupling, making it significant in thrombosis research and cardiovascular disease studies . The receptor is widely expressed across multiple tissue types, serving as a marker for Dorsal Excitatory Neurons in certain contexts .

What applications are FITC-conjugated P2RY1 antibodies suitable for?

FITC-conjugated P2RY1 antibodies are primarily optimized for:

• Flow cytometry (FCM)

• Immunofluorescence (IF)

• Western blotting (WB)

• Immunohistochemistry on paraffin-embedded tissues (IHC-p)

These conjugated antibodies eliminate the need for secondary antibody incubation steps, reducing background and cross-reactivity issues while enabling direct visualization of the target protein through fluorescence microscopy or flow cytometric analysis .

How do FITC-conjugated P2RY1 antibodies compare to unconjugated versions?

What is the typical reactivity profile of P2RY1 antibodies?

P2RY1 antibodies commonly demonstrate cross-reactivity across human, mouse, and rat species, reflecting the high conservation of this receptor across mammals. When analyzing Western blots, researchers should anticipate bands at approximately 42 kDa (theoretical weight), though observed molecular weights often range between 47-66 kDa due to post-translational modifications, particularly glycosylation .

The antibody raised against the human EL2 sequence exhibits significant interspecies reactivity and can recognize both denatured and native forms of human and mouse P2RY1 .

What are the optimal fixation and permeabilization protocols for FITC-conjugated P2RY1 antibodies?

For optimal results with FITC-conjugated P2RY1 antibodies in cellular applications:

Flow Cytometry:

• Fix cells with 2-4% paraformaldehyde for 10-15 minutes at room temperature

• Wash cells twice with PBS containing 1% BSA

• For intracellular detection, permeabilize with 0.1% Triton X-100 or 0.5% saponin for 5-10 minutes

• Incubate with FITC-conjugated P2RY1 antibody (typically at 1:50-1:100 dilution) for 30-60 minutes at room temperature in the dark

• Wash twice before analysis

Immunofluorescence:

• Fix cells/tissues with 4% paraformaldehyde for 15-20 minutes

• For membrane protein preservation, avoid methanol fixation which can disrupt the native conformation of membrane proteins like P2RY1

• Permeabilize with 0.1-0.2% Triton X-100 for 5-10 minutes

• Block with 5% normal serum for 30-60 minutes

• Apply FITC-conjugated P2RY1 antibody (1:50-1:200) for 1-2 hours or overnight at 4°C

• Counterstain nuclei with DAPI after thorough washing

How should platelets be prepared for P2RY1 antibody analysis?

Platelet preparation for P2RY1 analysis requires careful handling to prevent activation:

• Collect blood in acid-citrate-dextrose (ACD) anticoagulant (1:9 ratio)

• Obtain platelet-rich plasma by centrifugation at 200g for 20 minutes

• To prepare washed platelets:

  • Add 1μM PGE₁ to prevent activation during processing

  • Centrifuge at 800g for 10 minutes

  • Resuspend pellet in Tyrode's buffer (pH 7.4) containing 0.1% BSA

  • Adjust concentration to 2-3×10⁸ platelets/mL for flow cytometry

For antibody labeling, use gentle fixation (1% paraformaldehyde) for 10 minutes prior to antibody incubation. When examining P2RY1 receptor function, maintain platelets in a resting state until experimental activation is required .

What controls should be included when using FITC-conjugated P2RY1 antibodies?

A comprehensive control panel for FITC-conjugated P2RY1 antibody experiments should include:

Essential Controls:

• Isotype control: FITC-conjugated IgG of the same isotype, species, and concentration

• Unstained cells/tissues

• Secondary antibody-only control (for protocols using additional detection steps)

• Positive control: Cell line or tissue known to express P2RY1 (Jurkat cells or mouse heart tissue are recommended)

• Negative control: When possible, P2RY1 knockout samples or cells with validated absence of the target

• Peptide competition control: Pre-absorption of the antibody with its cognate peptide (EL2; 100 μM) to confirm specificity

For flow cytometry specifically, include single-color controls for compensation when performing multicolor analysis and use validated gating strategies based on forward/side scatter properties .

How can P2RY1 receptor internalization affect antibody detection?

P2RY1 receptor undergoes agonist-induced internalization, which can significantly impact antibody detection results. When activated by ADP or other agonists, P2RY1 receptors rapidly redistribute from the plasma membrane to intracellular compartments through clathrin-dependent endocytosis.

Methodological Considerations:

• For surface detection: Analyze samples within 15-30 minutes of isolation to minimize spontaneous internalization

• To capture total receptor pool: Include permeabilization steps in your protocol

• For internalization studies: Use pulse-chase approaches with FITC-conjugated antibodies

• Temperature control is critical: Maintain samples at 4°C to minimize receptor trafficking when measuring surface expression

• To distinguish surface from internalized receptors: Consider using acid wash protocols (pH 2.5) to strip surface-bound antibodies

Researchers studying receptor dynamics should consider time-course experiments to track P2RY1 movement between membrane and cytoplasmic compartments following stimulation with agonists like ADP .

How do glycosylation patterns affect P2RY1 antibody recognition?

P2RY1 undergoes significant post-translational modifications, particularly N-linked glycosylation, which explains the variable molecular weights observed in Western blots (ranging from 42-66 kDa) . These glycosylation patterns can affect antibody binding in several ways:

Impact on Antibody Recognition:

• Glycosylation may mask epitopes, particularly in the extracellular domains

• Different tissue sources may show variable glycosylation patterns, resulting in different apparent molecular weights

• Deglycosylation treatments (PNGase F or Endo H) can confirm antibody specificity by shifting band patterns

Experimental Approach for Resolving Glycosylation Variability:

• Run parallel samples with and without deglycosylation treatment

• Compare results across different tissue/cell types

• Use reducing and non-reducing conditions to evaluate potential effects of disulfide bonding

• When interpreting Western blot results showing multiple bands, consider that 42 kDa represents the core protein, while bands at 57-59 kDa and 66 kDa likely represent differentially glycosylated forms

What are the binding characteristics of anti-P2RY1 antibodies targeting the second extracellular loop?

Antibodies targeting the second extracellular loop (EL2) of P2RY1 possess unique functional characteristics due to their interaction with the receptor's ligand binding domain. These antibodies can:

• Compete directly with endogenous ligands for receptor binding

• Demonstrate function-blocking properties by preventing ADP-induced signaling

• Displace radiolabeled P2RY1 antagonists like [³H]MRS25000 in a dose-dependent manner

Research has shown that EL2-targeting antibodies inhibit multiple aspects of P2RY1-mediated platelet function, including:

• Integrin GPIIb-IIIa activation

• Dense and alpha granule secretion

• Phosphatidylserine exposure

• Thrombus formation in FeCl₃-induced thrombosis models

This makes EL2-targeting antibodies potentially valuable tools for both research and therapeutic applications. When using such antibodies, researchers should carefully titrate concentrations as competition with endogenous ligands follows dose-dependent kinetics .

How can background fluorescence be minimized when using FITC-conjugated P2RY1 antibodies?

High background is a common challenge with FITC-conjugated antibodies. To minimize non-specific signal:

Protocol Optimization Strategies:

• Increase blocking duration and concentration (use 5-10% serum from the same species as the secondary antibody)

• Include 0.1-0.3% Triton X-100 in blocking buffer to reduce hydrophobic interactions

• Add 0.05-0.1% Tween-20 to all wash buffers

• Utilize 1-2% BSA in dilution buffers to reduce non-specific binding

• Optimize antibody concentration through titration experiments (typical range 1:50-1:200)

• Include 10% normal serum from the host species in antibody dilution buffer

Tissue-Specific Considerations:

• For tissues with high autofluorescence (brain, liver, kidney), consider:

  • Brief treatment with 0.1% Sudan Black B in 70% ethanol after antibody incubation

  • Using lower exposure settings during image acquisition

  • Employing spectral unmixing during analysis

What strategies are effective when P2RY1 antibody shows weak or no signal?

When facing weak or absent signals with P2RY1 antibodies, consider these methodological approaches:

Signal Enhancement Methods:

• Antigen retrieval optimization:

  • For IHC applications: Test both citrate buffer (pH 6.0) and TE buffer (pH 9.0) to determine optimal conditions

  • Extend retrieval time to 20-30 minutes for difficult samples

• Antibody incubation modifications:

  • Increase concentration (use 1:20-1:200 for IHC applications)

  • Extend incubation time to overnight at 4°C

  • Add signal amplification steps (tyramide signal amplification or high-sensitivity detection systems)

• Sample-specific considerations:

  • Confirm target expression in your sample type (P2RY1 is detectable in Jurkat cells and mouse heart tissue)

  • Consider alternative detection methods (e.g., switch from IHC to Western blot)

  • Evaluate different sample preparation methods that better preserve membrane proteins

• Antibody quality control:

  • Test multiple antibody clones targeting different epitopes

  • Verify antibody functionality using positive control samples

  • Ensure proper antibody storage conditions

How can researchers validate P2RY1 antibody specificity in their experimental system?

Rigorous validation of P2RY1 antibody specificity is essential for reliable research outcomes:

Recommended Validation Approaches:

• Peptide Competition Assay:

  • Pre-incubate antibody with its cognate peptide (EL2; 100 μM)

  • Compare results with and without peptide competition

  • Specific binding should be substantially reduced or eliminated after peptide pre-absorption

• Genetic Controls:

  • Utilize P2RY1-knockout samples when available

  • Compare antibody reactivity in wild-type versus P2RY1 knockout tissues

  • Consider siRNA knockdown in cell culture systems

• Multi-technique Validation:

  • Confirm findings across multiple applications (WB, IHC, flow cytometry)

  • Compare results with different antibody clones targeting distinct epitopes

  • Verify that observed molecular weights match expected patterns (considering glycosylation)

• Functional Correlation:

  • Correlate antibody binding with functional assays (e.g., calcium mobilization assays)

  • For function-blocking antibodies, confirm inhibition of ADP-induced signaling

  • Demonstrate dose-dependent displacement of known P2RY1 ligands

How can FITC-conjugated P2RY1 antibodies be used in receptor internalization studies?

FITC-conjugated P2RY1 antibodies offer valuable tools for investigating receptor trafficking dynamics:

Experimental Design for Internalization Studies:

• Time-course visualization:

  • Pulse-label surface receptors at 4°C with FITC-conjugated P2RY1 antibody

  • Warm cells to 37°C in the presence of agonist (ADP)

  • Fix cells at defined time points (0, 5, 15, 30, 60 minutes)

  • Counterstain with membrane markers to quantify receptor internalization rate

• Flow cytometric quantification:

  • Label cells with FITC-conjugated P2RY1 antibody

  • Split samples for unstimulated vs. ADP-stimulated conditions

  • Measure decrease in surface fluorescence intensity following stimulation

  • To distinguish internalization from shedding, include permeabilized samples to quantify total receptor pool

• Advanced microscopy approaches:

  • Combine FITC-conjugated P2RY1 antibody with markers for endocytic compartments

  • Use live-cell imaging to track receptor movement in real-time

  • Employ pH-sensitive fluorescent proteins to distinguish surface from internalized receptors

  • Quantify co-localization with early endosome markers (EEA1) versus recycling compartments (Rab11)

What approaches can resolve contradictory findings in P2RY1 expression studies?

When researchers encounter contradictory results regarding P2RY1 expression patterns:

Reconciliation Strategies for Conflicting Data:

• Epitope accessibility considerations:

  • Different antibodies may target distinct epitopes with variable accessibility

  • Test multiple antibodies targeting different domains (N-terminal, C-terminal, extracellular loops)

  • Compare results from antibodies recognizing denatured versus native conformations

• Methodological differences:

  • Systematically compare fixation methods (aldehyde vs. organic solvent)

  • Evaluate different antigen retrieval approaches (pH 6.0 vs. pH 9.0)

  • Standardize tissue processing across laboratories

• Biological variability explanations:

  • Investigate receptor expression across different developmental stages

  • Compare healthy versus pathological tissues

  • Evaluate regulation under different physiological conditions

  • Consider splice variants and post-translational modifications

• Quantitative validation:

  • Complement protein detection with mRNA analysis (RT-qPCR)

  • Use absolute quantification methods where possible

  • Implement rigorous statistical analysis of expression patterns across multiple samples

How can researchers assess P2RY1 receptor functionality using antibody-based approaches?

Beyond simple detection, antibodies can provide insights into P2RY1 receptor functionality:

Functional Assessment Methodologies:

• Function-blocking assays:

  • Use EL2-targeting antibodies to inhibit ADP-induced platelet aggregation

  • Measure dose-dependent effects on:

    • Integrin GPIIb-IIIa activation

    • Dense and alpha granule secretion

    • Phosphatidylserine exposure

• Receptor-ligand interaction studies:

  • Employ competitive binding assays with radiolabeled antagonists like [³H]MRS25000

  • Measure displacement curves with EL2Ab antibodies

  • Determine binding constants and competition kinetics

• Signaling pathway analysis:

  • Correlate P2RY1 detection with downstream signaling events

  • Monitor calcium mobilization following receptor stimulation

  • Assess phosphorylation of signaling intermediates (PLCβ, PKC)

  • Examine effects of function-blocking antibodies on signaling cascades

• In vivo functional studies:

  • Evaluate the effects of antibodies in thrombosis models

  • Measure occlusion time in FeCl₃-induced vascular injury

  • Assess bleeding time in appropriate models

  • Compare antibody effects with known pharmacological antagonists

How does P2RY1 expression and detection differ in thrombotic disorders?

P2RY1 receptor plays a crucial role in platelet activation and thrombosis, making it relevant for studying various thrombotic disorders:

Expression Patterns and Detection Considerations:

For accurate detection in disease models, researchers should:

• Compare P2RY1 receptor density between patient and healthy control samples

• Correlate receptor expression with functional assays (ADP-induced aggregation)

• Consider using multiple detection methods (flow cytometry + Western blotting)

• Account for medication effects (P2Y₁₂ antagonists) on receptor regulation

What are the optimal protocols for multiplexed detection of P2RY1 with other platelet receptors?

Multiplexed detection of P2RY1 with other platelet receptors provides comprehensive insights into receptor complexes and signaling networks:

Optimized Multiplexing Strategies:

• Multicolor flow cytometry:

  • Combine FITC-conjugated P2RY1 antibody with spectrally distinct fluorochromes for other targets

  • Recommended combinations:

    • P2RY1-FITC + P2Y₁₂-PE + GPIIb/IIIa-APC

    • P2RY1-FITC + PAR1-PE + PAR4-APC

  • Include single-color controls for proper compensation

  • Use hierarchical gating strategies to identify platelet subpopulations

• Multiplex immunofluorescence imaging:

  • Sequential staining protocols with careful antibody stripping between rounds

  • Tyramide signal amplification for weak signals

  • Spectral unmixing to resolve overlapping fluorophores

  • Consider using quantum dots for multiplexed detection with minimal spectral overlap

• Proximity ligation assays:

  • Detect P2RY1 interactions with other receptors at molecular proximity (<40 nm)

  • Evaluate receptor dimerization or complex formation

  • Visualize receptor clustering following activation

• Mass cytometry considerations:

  • Metal-conjugated antibodies for high-dimensional analysis

  • Simultaneous detection of >30 parameters

  • Ideal for comprehensive platelet receptor profiling

How can researchers quantitatively analyze P2RY1 receptor expression in tissue microarrays?

Tissue microarrays (TMAs) enable high-throughput analysis of P2RY1 expression across multiple samples, requiring robust quantification approaches:

Quantitative Analysis Protocols:

• Staining optimization for TMAs:

  • Standardize fixation and processing across all TMA cores

  • Implement automated staining platforms for consistency

  • Include control tissues (positive and negative) on each TMA

  • Use FITC-conjugated P2RY1 antibody at validated dilutions (typically 1:50-1:100)

• Image acquisition parameters:

  • Standardize exposure settings across all samples

  • Capture multiple fields per core when expression is heterogeneous

  • Use automated microscopy for consistent imaging

  • Include fluorescence calibration standards

• Quantification methodologies:

  • Membrane-specific segmentation algorithms

  • Intensity thresholding based on control samples

  • Quantify parameters including:

    • Mean fluorescence intensity

    • Percentage of positive cells

    • Membrane-to-cytoplasm signal ratio

    • Expression heterogeneity metrics

• Data analysis approaches:

  • Normalize expression against housekeeping proteins

  • Implement machine learning classification of expression patterns

  • Correlate expression with clinical parameters

  • Use hierarchical clustering to identify tissue-specific expression patterns