P2RX7 Antibody, Biotin conjugated

What is the P2X7 receptor and what roles does it play in cellular function?

The P2X7 receptor (P2RX7) is a ligand-gated ion channel activated by extracellular ATP that functions in multiple critical cellular processes. It is responsible for ATP-dependent lysis of macrophages through the formation of membrane pores permeable to large molecules . Research has demonstrated that P2X7 also acts as a scavenger receptor for apoptotic cells in its unactivated state, participating in phagocytosis of apoptotic lymphocytes and neuronal cells by macrophages under serum-free conditions . Additionally, P2X7 plays regulatory roles in immune responses, particularly in T follicular helper (Tfh) cell expansion during autoimmune conditions .

What are the advantages of using biotin-conjugated P2RX7 antibodies in experimental workflows?

Biotin-conjugated P2RX7 antibodies offer significant experimental advantages:

• Enhanced detection sensitivity through the high-affinity biotin-streptavidin interaction

• Experimental flexibility through compatibility with various streptavidin-conjugated detection systems

• Signal amplification capabilities for detecting low-abundance receptors

• Integration with multiple analytical platforms including flow cytometry, immunohistochemistry, and ELISA

• Reduced background in tissues with high endogenous peroxidase or phosphatase activity

The biotin conjugation also allows for multi-parameter studies when using fluorescently labeled streptavidin conjugates, enabling correlation between P2X7 expression and functional outcomes .

What methodological approaches can verify P2RX7 antibody specificity?

Verifying antibody specificity is critical for reliable P2X7 research. Recommended approaches include:

• Western blot analysis comparing P2X7-expressing tissues (brain, macrophages) with appropriate controls

• Testing the antibody on cells from P2X7 knockout (P2X7−/−) mice as negative controls

• Flow cytometric analysis comparing cells transfected with human P2X7 versus other P2X receptor types

• Pre-absorption tests with the immunizing peptide to confirm binding specificity

• Immunoprecipitation followed by mass spectrometry to identify pulled-down proteins

Research has demonstrated that properly validated P2RX7 antibodies show specific labeling of human blood-derived macrophages natively expressing P2X7 receptors and cells transfected with human P2X7 but not other P2X receptor types .

How do extracellular cysteine residues in P2X7 contribute to its role in phagocytosis of apoptotic cells?

The extracellular domain of P2X7 contains critical cysteine residues that directly participate in apoptotic cell recognition. Research using a peptide screen library with 24 biotin-conjugated peptides mimicking the extracellular domain of P2X7 revealed:

• Cysteine-containing peptides uniquely bind to apoptotic cell surfaces but not to viable cells

• Substitution of cysteine with alanine in these peptides abolishes binding

• Thiol-reactive compounds including N-acetyl-L-cysteine inhibit phagocytosis of apoptotic cells

These findings indicate that extracellular disulfide bonds in the P2X7 receptor play a crucial role in the direct recognition and engulfment of apoptotic cells, supporting P2X7's function as a scavenger receptor in its unactivated state .

What is the role of P2X7 in autoimmune disease pathogenesis and how can biotin-conjugated antibodies help investigate this?

P2X7 exhibits complex regulatory functions in autoimmune diseases like systemic lupus erythematosus (SLE). Research shows that P2X7 selectively limits potentially pathogenic T follicular helper (Tfh) cell expansion:

• P2rx7−/− mice treated with pristane (to induce lupus-like disease) show enhanced secretion of self-reactive antibodies

• These mice exhibit increased IgG production, particularly IgG1 and IgG2B subtypes

• P2rx7−/− mice develop abnormal germinal center reactions with elevated autoantibody production

• The effect is specific to autoreactive responses, as P2rx7−/− mice show impaired responses to conventional immunization

Biotin-conjugated P2RX7 antibodies enable precise tracking of receptor expression in immune cell subsets via multiparameter flow cytometry, allowing correlation of receptor levels with functional outcomes in autoimmune contexts .

How does P2X7 receptor expression and function differ across tissue microenvironments?

P2X7 receptor expression and function show significant tissue-specific modulation:

Research demonstrates that P2X7 surface expression on phagocytes increases significantly during phagocytosis of beads or apoptotic cells . The receptor's function is modulated by local microenvironmental factors including pH, divalent cation concentration, and exposure to specific antagonists like Brilliant Blue G .

What are optimal protocols for using biotin-conjugated P2RX7 antibodies in flow cytometry?

For effective flow cytometry using biotin-conjugated P2RX7 antibodies:

• Cell preparation:

  • For suspended cells: Use 0.50 μg antibody per 10^6 cells in 100 μl buffer

  • For adherent cells: Gentle enzymatic detachment preferred over mechanical methods

  • Fresh isolation yields better results than frozen samples

• Staining protocol:

  • For intracellular staining: Fix with 4% paraformaldehyde, permeabilize with 0.1% saponin

  • Block with 5% serum in PBS for 20 minutes

  • Incubate with biotin-conjugated P2RX7 antibody (1:100-1:500 dilution)

  • After washing, incubate with streptavidin-fluorophore conjugate

  • Include proper controls: isotype control, unstained cells, and when possible, P2X7−/− samples

• Analysis considerations:

  • Gate based on forward/side scatter to exclude debris and dead cells

  • Titrate antibody concentration for optimal signal-to-noise ratio

  • Consider functional correlation using ATP-induced dye uptake assays

How can I optimize Western blot protocols for P2RX7 detection?

Optimized Western blot protocol for P2RX7 detection:

• Sample preparation:

  • Extract proteins using buffer containing protease inhibitors

  • Heat samples to 98°C for 5 minutes in sample buffer

  • Load 80 μg total protein per lane

• Electrophoresis and transfer:

  • Use 10% SDS-PAGE gel for optimal separation

  • Transfer to PVDF membrane (Immobilon-P recommended)

• Blocking and antibody incubation:

  • Block with 5% fat-free milk and 0.2% Tween 20 in TBS for 2 hours

  • Incubate with primary antibody (1:500-1:3000 dilution) overnight at 4°C

  • Use HRP-conjugated secondary antibody at 1:1000 dilution

• Detection and troubleshooting:

  • Visualize using chemiluminescent detection systems like ECL Plus

  • Expected molecular weight: 70-80 kDa

  • Include positive controls (mouse brain tissue, cerebellum tissue)

  • For negative control, use antibody pre-absorbed with control peptide

What experimental design is appropriate for studying P2X7-mediated phagocytosis?

To effectively study P2X7-mediated phagocytosis:

• In vitro model system:

  • Human monocyte-derived macrophages cultured in serum-free conditions

  • Fluorescently labeled apoptotic cells (lymphocytes or neuronal cells)

  • Comparative analysis with established phagocytosis inhibitors (cytochalasin D)

  • P2X7 antagonist (ATP) as competitive inhibitor

• In vivo approach:

  • Injection of apoptotic thymocytes into mouse peritoneal cavity

  • Pre-treatment with ATP to inhibit P2X7-mediated phagocytosis

  • Comparative analysis between wild-type and P2X7-deficient mice

  • Flow cytometric quantification of phagocytosis

• Mechanistic investigation:

  • Biotin-conjugated peptides mimicking P2X7 extracellular domains

  • Evaluation of binding profiles to apoptotic cells versus viable cells

  • Substitution studies (cysteine to alanine) to identify critical residues

  • Thiol-reactive compound (N-acetyl-L-cysteine) effects on phagocytosis

How can I differentiate between specific and non-specific signals when using biotin-conjugated antibodies?

Differentiating specific from non-specific signals requires systematic controls:

• Negative controls:

  • Sections incubated with PBS alone instead of primary antibody

  • Isotype control antibodies at equivalent concentration

  • Pre-absorption of antibody with immunizing peptide

  • Samples from P2X7−/− mice or knockdown cell lines

• Addressing biotin-specific challenges:

  • Block endogenous biotin using avidin/biotin blocking kits

  • Use streptavidin-conjugates with different detection properties to confirm signal pattern

  • Include biotin-conjugated non-specific antibody as control

• Signal validation approaches:

  • Compare results across multiple detection methods (flow cytometry, immunohistochemistry, Western blot)

  • Verify antibody specificity using cells transfected with P2X7 versus other P2X receptor types

  • Confirm correct molecular weight (70-80 kDa) in Western blot analyses

What factors affect P2X7 detection in different experimental systems?

Multiple factors influence P2X7 detection across experimental platforms:

Research demonstrates that P2X7 surface expression on phagocytes increases significantly during functional activity, which may affect detection depending on cellular state .

How can I validate unexpected P2X7 expression patterns observed with biotin-conjugated antibodies?

When confronted with unexpected P2X7 expression patterns:

• Technical validation:

  • Repeat experiments with different antibody concentrations and detection systems

  • Compare results with antibodies targeting different P2X7 epitopes

  • Use multiple detection methods (Western blot, flow cytometry, immunohistochemistry)

• Biological validation:

  • Correlate expression with functional assays (ATP-induced pore formation)

  • Verify findings using genetic approaches (siRNA knockdown, CRISPR-Cas9)

  • Test in P2X7-deficient systems as negative controls

• Context considerations:

  • Evaluate potential splice variants (Western blot may show multiple bands)

  • Consider species differences if comparing across model systems

  • Examine whether experimental conditions (activation state, cytokines) alter P2X7 expression

  • Evaluate serum-dependent versus serum-free conditions, which significantly affect P2X7's functional role

How do I interpret changes in P2X7 expression in disease models?

Interpreting P2X7 expression changes in disease contexts requires:

• Comparative analysis:

  • Quantify expression levels relative to appropriate controls

  • Compare multiple detection methods to confirm changes

  • Consider both protein and mRNA expression patterns

• Functional correlation:

  • Assess P2X7-dependent functions (pore formation, phagocytosis)

  • In autoimmune contexts, examine relationship between P2X7 expression and disease parameters

  • Consider T follicular helper cell development and germinal center reactions in P2rx7−/− models

• Contextual interpretation:

  • In autoimmune diseases like SLE, decreased P2X7 function correlates with enhanced autoantibody production

  • P2rx7−/− mice show increased susceptibility to pristane-induced lupus but impaired responses to conventional immunization

  • Changes may be cell-type specific (macrophages vs. T cells)

  • Expression changes may reflect altered cell populations rather than per-cell expression differences