P2RY1 Antibody, Biotin conjugated

What is P2RY1 and why is it an important research target?

P2RY1 (purinergic receptor P2Y, G-protein coupled, 1) is a G-protein coupled receptor expressed in the cell membrane that functions as a receptor for extracellular adenine nucleotides, particularly ADP . With a canonical molecular weight of approximately 42 kDa (though observed weights can vary from 42-66 kDa due to post-translational modifications like glycosylation), P2RY1 is widely expressed across multiple tissue types . It serves as an important marker for identifying Dorsal Excitatory Neurons and has been implicated in various physiological processes including platelet aggregation, neuronal signaling, and vascular responses . The receptor is also known by several synonyms including SARCC, P2Y purinoceptor 1, ADP receptor, ATP receptor, and P2 purinoceptor subtype Y1 .

What are the advantages of using biotin-conjugated P2RY1 antibodies?

Biotin-conjugated P2RY1 antibodies offer several methodological advantages in immunodetection protocols. The biotin-streptavidin system provides significant signal amplification due to the high affinity between biotin and streptavidin (Kd ≈ 10^-15 M), enabling enhanced sensitivity in detection systems . This conjugation allows for versatile secondary detection using various streptavidin-conjugated reporter molecules (fluorophores, enzymes, quantum dots), providing flexibility in experimental design . Additionally, the small size of biotin molecules minimizes steric hindrance that might otherwise interfere with antibody-antigen binding, preserving the specificity and affinity of the primary antibody .

How should biotin-conjugated P2RY1 antibodies be stored for optimal stability?

For long-term storage, biotin-conjugated P2RY1 antibodies should be stored at -20°C, where they typically remain stable for up to one year . For frequent use and short-term storage (up to one month), 4°C is recommended to minimize freeze-thaw cycles that can degrade antibody quality . Most commercially available preparations come in buffer solutions containing stabilizers such as glycerol (often at 50%) and preservatives like sodium azide (0.02%) or Proclin-300 (0.03%) . It's crucial to aliquot the antibody upon receipt to avoid repeated freeze-thaw cycles, and exposure to light should be minimized, particularly for detection strategies involving fluorescence measurements .

What are the optimal dilutions for biotin-conjugated P2RY1 antibodies in different applications?

The optimal dilution of biotin-conjugated P2RY1 antibodies varies by application technique and specific antibody preparation. For Western blotting applications, dilutions typically range from 1:500-1:1000 . For immunohistochemistry protocols, a broader range of 1:20-1:200 may be necessary, with optimal conditions determined empirically . For ELISA applications, the dilution is often antibody-specific, and manufacturers generally recommend that end users determine optimal concentrations through titration experiments . Flow cytometry applications typically require dilutions specified by the manufacturer's protocol, often expressed as a volume per test (e.g., 5-10 μL per test) . It's important to note that these values should serve as starting points, and optimal conditions should be established for each specific experimental system.

What antigen retrieval methods are recommended for IHC applications with P2RY1 antibodies?

For immunohistochemical detection of P2RY1, optimal antigen retrieval methods typically involve heat-induced epitope retrieval (HIER) using either TE buffer at pH 9.0 or citrate buffer at pH 6.0 . The TE buffer (pH 9.0) method is generally preferred as the primary recommendation, with citrate buffer serving as an alternative approach when results are suboptimal . For formalin-fixed, paraffin-embedded tissues, retrieval typically involves heating the sections in the appropriate buffer for 15-20 minutes at 95-100°C, followed by cooling to room temperature before proceeding with immunostaining protocols. This step is critical for breaking protein cross-links formed during fixation, thereby exposing epitopes for antibody binding and improving detection sensitivity.

How can I verify the specificity of a biotin-conjugated P2RY1 antibody in my experimental system?

Verifying antibody specificity requires a multi-faceted approach. Begin with positive and negative control tissues with known P2RY1 expression patterns; human testis, kidney, lung, ovary, and placenta tissues have shown positive immunoreactivity for P2RY1 . For cellular systems, Jurkat cells and mouse heart tissue have demonstrated detectable P2RY1 expression in Western blotting applications .

Include appropriate negative controls in your experiments, such as:

• Isotype controls using non-specific biotin-conjugated antibodies of the same isotype

• Blocking peptide competition assays, where pre-incubation with the immunogenic peptide should abolish specific staining

• Testing in tissues or cell lines with P2RY1 gene knockdown or knockout

Additionally, compare the observed molecular weight with the expected range (canonical 42 kDa, with observations from 42-66 kDa depending on post-translational modifications) . For biotin-conjugated antibodies specifically, include a streptavidin-only control to assess potential endogenous biotin interference in your experimental system.

How can P2RY1 antibodies be used to investigate purinergic signaling in different neural populations?

P2RY1 serves as a marker for Dorsal Excitatory Neurons, making it valuable for neurological research . When investigating purinergic signaling in neural populations, researchers can employ multi-parameter flow cytometry using biotin-conjugated P2RY1 antibodies combined with streptavidin-fluorophore conjugates of distinct emission spectra alongside other neural markers for comprehensive phenotyping .

For brain slice or tissue section analysis, immunohistochemistry with P2RY1 antibodies can be combined with electrophysiological recording techniques to correlate purinergic receptor expression with functional responses to ATP/ADP stimulation . In primary neuronal cultures, biotin-conjugated P2RY1 antibodies can be used in calcium imaging experiments to identify cells responding to purinergic stimulation through changes in intracellular calcium levels.

Research protocols should include:

• Parallel visualization of downstream signaling molecules (e.g., calcium imaging with Fluo-4)

• Co-localization studies with other purinergic receptors to map receptor distribution patterns

• Functional assays combining P2RY1 immunostaining with electrophysiological recordings

What approaches can resolve the discrepancy between predicted and observed molecular weights of P2RY1 in Western blotting?

The discrepancy between the predicted molecular weight of P2RY1 (42 kDa) and the observed weights (ranging from 42-66 kDa) in Western blotting is a common research challenge . To resolve this discrepancy and ensure accurate identification, researchers should implement a systematic analytical approach:

• Enzymatic deglycosylation: Treat samples with PNGase F or other glycosidases before Western blotting to remove N-linked glycans, which should reduce higher molecular weight bands if glycosylation is responsible for the apparent size difference

• Phosphatase treatment: Incubate samples with lambda phosphatase to eliminate phosphorylation-induced mobility shifts

• Sample preparation variations:

  • Compare reducing vs. non-reducing conditions

  • Test different lysis buffers to assess impact of detergent type on apparent molecular weight

  • Vary sample heating conditions (37°C vs. 95°C) to assess effects on protein conformation

• Verification methods:

  • Immunoprecipitation followed by mass spectrometry analysis to confirm identity

  • RNA interference to confirm band specificity by demonstrating reduced intensity after P2RY1 knockdown

  • Compare with recombinant P2RY1 protein standards run on the same gel

The presence of multiple bands may represent different isoforms, post-translational modifications, or proteolytic fragments of P2RY1, rather than non-specific binding .

How can biotin-conjugated P2RY1 antibodies be used effectively in multiplex immunoassays with tissues showing high endogenous biotin?

Some tissues (brain, kidney, liver) contain high levels of endogenous biotin that can cause background signal and false positives when using biotin-conjugated antibodies. To overcome this challenge in multiplex immunoassays, researchers should implement a strategic approach:

• Endogenous biotin blocking protocol:

  • Pre-treat tissue sections with avidin/biotin blocking kit

  • Incubate sections with free streptavidin (10-20 μg/mL) for 15 minutes

  • Follow with excess free biotin (50-100 μg/mL) for 15 minutes before applying the biotin-conjugated primary antibody

• Alternative detection strategy:

  • Use a directly labeled secondary antibody against the host species of the P2RY1 antibody

  • Select a fluorophore or enzyme with a spectrally distinct signature from other detection reagents in the multiplex assay

• Sequential multiplex protocol:

  • Apply and detect biotin-conjugated P2RY1 antibody first

  • Follow with a biotin-quenching step using excess free streptavidin

  • Proceed with subsequent non-biotin antibodies and their corresponding detection systems

• Control experiments:

  • Include a streptavidin-only control section to assess baseline endogenous biotin levels

  • Consider using a non-biotinylated P2RY1 antibody as a comparative control

This systematic approach ensures robust and specific detection of P2RY1 even in tissues with high endogenous biotin content, while maintaining the ability to perform multiplex analysis with additional markers.

What are the common causes of high background when using biotin-conjugated P2RY1 antibodies, and how can they be addressed?

High background signal is a common challenge when working with biotin-conjugated antibodies, including those targeting P2RY1. The following table outlines common causes and their solutions:

When optimizing your protocol, change only one parameter at a time to systematically identify the source of background issues .

How can I distinguish between specific and non-specific binding when using P2RY1 antibodies in Western blotting?

Distinguishing between specific and non-specific binding is crucial for accurate interpretation of Western blotting results with P2RY1 antibodies. A systematic validation approach includes:

• Molecular weight verification:

  • P2RY1 typically appears at 42 kDa (canonical) but can range from 42-66 kDa due to post-translational modifications

  • Compare observed bands with recombinant P2RY1 protein control run on the same gel

  • Note that multiple specific bands may represent different glycosylation states or isoforms

• Validation controls:

  • Peptide competition assay: Pre-incubation of antibody with immunizing peptide should abolish specific bands

  • Knockdown/knockout validation: Samples from P2RY1 knockdown or knockout systems should show reduced or absent specific bands

  • Positive control samples: Include known P2RY1-expressing samples (e.g., Jurkat cells, mouse heart tissue)

• Protocol optimization:

  • Titrate primary antibody concentration (typical range: 1:500-1:1000)

  • Optimize blocking conditions using 5% non-fat milk or BSA

  • Increase washing stringency with higher salt concentration or detergent (0.1-0.3% Tween-20)

• Advanced verification techniques:

  • Use two different P2RY1 antibodies recognizing distinct epitopes

  • Immunoprecipitation followed by Western blotting

  • Mass spectrometry confirmation of band identity

A band is likely specific if it: appears at the expected molecular weight, diminishes with peptide competition, reduces in knockdown samples, and is consistent across different antibodies targeting the same protein.

How should flow cytometry data be analyzed when examining P2RY1 expression in heterogeneous cell populations?

Flow cytometry analysis of P2RY1 expression in heterogeneous cell populations requires a systematic gating strategy and appropriate controls:

• Gating hierarchy:

  • Begin with time gate to exclude acquisition artifacts

  • Apply FSC/SSC gating to identify intact cells and remove debris

  • Use viability dye to exclude dead cells (critical for accurate P2RY1 assessment)

  • Apply lineage markers to identify specific cell populations of interest

  • Finally, analyze P2RY1 expression within defined subpopulations

• Essential controls:

  • Fluorescence Minus One (FMO) control: Include all fluorochromes except the one conjugated to streptavidin for detecting biotinylated P2RY1 antibody

  • Isotype control: Use biotin-conjugated isotype-matched irrelevant antibody

  • Biological controls: Include known P2RY1 positive and negative cell populations

• Data presentation and analysis:

  • Report P2RY1 expression as percent positive cells and median fluorescence intensity (MFI)

  • Use fold change in MFI relative to control for quantitative comparisons

  • For heterogeneous populations, present data as bivariate plots showing P2RY1 versus relevant lineage markers

• Advanced analysis techniques:

  • Consider visualization tools like t-SNE or UMAP for high-dimensional data analysis

  • Apply hierarchical clustering to identify novel P2RY1-expressing subpopulations

  • Use Boolean gating to characterize cells co-expressing P2RY1 with other markers

When analyzing samples with potential endogenous biotin (e.g., tissue-derived cells), include a streptavidin-only control to establish the background signal threshold .

What are the emerging applications of P2RY1 antibodies in neurodegenerative disease research?

P2RY1 antibodies are increasingly being utilized in neurodegenerative disease research, reflecting the growing recognition of purinergic signaling's role in neurological conditions. Recent studies have employed these antibodies to:

• Characterize purinergic receptor redistribution in Alzheimer's disease models, where P2RY1 expression patterns may change in response to amyloid-β accumulation

• Investigate neuroinflammatory processes in Parkinson's disease, with P2RY1-positive astrocytes showing altered reactivity states that may contribute to disease progression

• Examine the role of P2RY1 in astrocyte-mediated neurotoxicity in ALS models, where aberrant calcium signaling through purinergic receptors may influence disease course

• Study potential neuroprotective mechanisms in stroke models, where P2RY1 activation has shown context-dependent effects on neuronal survival

Methodologically, researchers are employing multiplexed immunofluorescence approaches using biotin-conjugated P2RY1 antibodies alongside markers for cellular stress, neuroinflammation, and synaptic integrity to create comprehensive spatial profiles of purinergic signaling alterations in disease states . The biotin conjugation facilitates signal amplification strategies that are particularly valuable when working with human post-mortem tissue samples where antigen preservation may be suboptimal.

How can contradictory findings about P2RY1 expression patterns across different studies be reconciled?

Contradictory findings regarding P2RY1 expression patterns across different studies can be reconciled through a systematic analytical approach:

• Methodological differences assessment:

  • Antibody variability: Different antibodies may recognize distinct epitopes or isoforms of P2RY1

  • Fixation protocols: Overfixation can mask epitopes, while inadequate fixation may alter tissue morphology

  • Detection systems: Sensitivity differences between chromogenic and fluorescent detection methods

  • Antigen retrieval methods: Different buffers and pH conditions affect epitope accessibility

• Sample-specific considerations:

  • Developmental stage: P2RY1 expression may vary temporally during development

  • Species differences: Human vs. rodent P2RY1 expression patterns may differ

  • Disease state: Pathological conditions may alter receptor expression or localization

  • Regional variations: Expression heterogeneity within tissues requires comprehensive sampling

• Reconciliation strategies:

  • Meta-analysis of multiple studies with attention to methodological details

  • Side-by-side comparison using multiple antibodies on the same samples

  • Correlation of protein expression with mRNA data (ISH, RNA-seq)

  • Functional validation through calcium imaging or other purinergic signaling assays

• Reporting standards:

  • Detailed documentation of antibody validation procedures

  • Clear description of tissue processing and staining protocols

  • Quantitative assessment using digital image analysis

  • Transparent presentation of both positive and negative findings

By addressing these factors systematically, researchers can better understand whether contradictory findings reflect biological reality or technical artifacts .

What innovative approaches are being developed to improve the specificity and sensitivity of P2RY1 detection in complex tissue samples?

Several innovative approaches are emerging to enhance the specificity and sensitivity of P2RY1 detection in complex tissue samples:

• Proximity ligation assay (PLA) adaptations:

  • Combining biotin-conjugated P2RY1 antibodies with antibodies against known interacting partners

  • This approach generates fluorescent signals only when P2RY1 and its partner are in close proximity (<40 nm)

  • Provides superior specificity by requiring coincident detection of multiple epitopes

• Expansion microscopy integration:

  • Physical expansion of tissue samples (4-10×) using hydrogel embedding

  • Combined with biotin-conjugated P2RY1 antibodies and fluorescent streptavidin detection

  • Enables super-resolution imaging of P2RY1 distribution using standard confocal microscopy

• CODEX (CO-Detection by indEXing) multiplexing:

  • Sequential detection of multiple biomarkers including P2RY1 on the same tissue section

  • Uses DNA-barcoded antibodies and complementary fluorescent oligonucleotides

  • Allows visualization of P2RY1 in relation to dozens of other markers in the same sample

• Advanced image analysis algorithms:

  • Machine learning approaches for automated P2RY1 detection and quantification

  • Deep learning neural networks trained to distinguish specific from non-specific staining patterns

  • Computational removal of autofluorescence through spectral unmixing algorithms

• Single-cell proteogenomic correlation:

  • Integration of P2RY1 protein detection with single-cell RNA sequencing

  • Validates antibody specificity through correlation of protein and mRNA expression

  • Provides comprehensive cellular context for P2RY1 expression patterns

These approaches collectively address the traditional limitations of antibody-based detection by combining multiple validation layers and leveraging technological advances in imaging and computational analysis .