P2RX4 Antibody

What is P2RX4 and why is it significant in scientific research?

P2RX4 (purinergic receptor P2X, ligand-gated ion channel, 4) is an ATP-gated nonselective transmembrane cation channel permeable to potassium, sodium, and calcium. This receptor plays multiple crucial roles in immunity and central nervous system physiology. With a molecular weight of approximately 43.4 kDa, P2RX4 is involved in initial steps of T-cell activation and Ca²⁺ microdomain formation. The receptor promotes the differentiation and activation of Th17 cells via expression of retinoic acid-related orphan receptor C/RORC and, upon activation, drives microglia motility via the PI3K/Akt pathway. P2RX4 may also function as an ATP-gated cation channel of lysosomal membranes, making it a significant target for research across neuroscience, immunology, and cell biology fields .

What are the key differences between polyclonal and monoclonal P2RX4 antibodies in research applications?

Polyclonal P2RX4 antibodies (like Proteintech's 13534-1-AP) recognize multiple epitopes on the P2RX4 protein, offering high sensitivity and robust signal in applications like Western blot, but with potential for increased background. These antibodies are typically produced in rabbit hosts and purified through antigen affinity methods . In contrast, monoclonal P2RX4 antibodies (such as Proteintech's 66416-1-Ig) recognize a single epitope, providing higher specificity but potentially lower sensitivity in certain applications. The monoclonal antibodies are frequently mouse-derived and purified using Protein A . Western blot validation shows polyclonal antibodies typically detect P2RX4 at 45-60 kDa, while monoclonals more consistently detect it at 50 kDa. For applications requiring extensive cross-species reactivity, polyclonal antibodies often demonstrate broader reactivity profiles across human, mouse, rat, and other species .

How should P2RX4 antibodies be stored and handled to maintain optimal reactivity?

P2RX4 antibodies require specific storage conditions to preserve their functionality. Most commercial P2RX4 antibodies should be stored at -20°C where they remain stable for one year after shipment. Many are supplied in PBS buffer containing 0.02% sodium azide and 50% glycerol at pH 7.3 to prevent freeze-thaw damage . When working with lyophilized antibody preparations, proper reconstitution is critical—typically adding 0.2 ml of distilled water to yield a concentration of 500 μg/ml . After reconstitution, short-term storage at 4°C is suitable for up to one month, but for longer preservation, aliquoting and storage at -20°C for up to six months is recommended. Repeated freeze-thaw cycles should be avoided as they progressively degrade antibody quality. Some antibody preparations may contain small amounts (0.1%) of BSA as a stabilizer, which should be noted when designing experiments .

What criteria should researchers use when selecting a P2RX4 antibody for specific experimental applications?

Selection of appropriate P2RX4 antibodies should be guided by several critical criteria. First, consider the intended application—different antibodies demonstrate variable performance across Western blotting, immunohistochemistry, immunofluorescence, flow cytometry, and ELISA applications. For instance, Proteintech's 13534-1-AP has been validated for WB, IHC, IF, and ELISA, while some antibodies like Creative Biolabs' TAB-0914CL are optimized specifically for ELISA . Second, evaluate species reactivity requirements—while some antibodies react only with human P2RX4 (such as Boster Bio's A04715-2), others show cross-reactivity with multiple species including mouse, rat, rabbit, and pig samples (like Proteintech's 66416-1-Ig) . Third, assess the immunogen used for antibody production—antibodies raised against different regions of P2RX4 (N-terminal versus full-length protein) may perform differently in detecting native versus denatured protein. Finally, consider validation evidence provided by manufacturers, including knockout/knockdown controls, which offers the most reliable confirmation of specificity .

How can researchers validate the specificity of P2RX4 antibodies to ensure reliable experimental results?

Validation of P2RX4 antibody specificity requires a multi-faceted approach. The gold standard involves using knockout/knockdown controls, comparing antibody reactivity in wild-type samples versus those with reduced or eliminated P2RX4 expression. Several published studies have utilized this approach, with 13534-1-AP antibody having cited knockout validation in the literature . Additionally, researchers should verify the detected molecular weight matches the expected size of P2RX4 (approximately 43 kDa theoretical weight, though observed weights range from 45-60 kDa depending on post-translational modifications and glycosylation) . Cross-reactivity testing with related P2X family proteins can further confirm specificity. For tissue-specific applications, comparative analysis of P2RX4 expression patterns with published data provides additional validation. Researchers should also consider performing peptide competition assays, where pre-incubation of the antibody with the immunizing peptide should abolish specific binding. Finally, using multiple antibodies targeting different epitopes of P2RX4 and observing consistent staining patterns provides strong evidence for specificity .

What are the optimal dilution ranges for P2RX4 antibodies in different experimental applications?

The optimal dilution ranges for P2RX4 antibodies vary significantly by application and specific antibody product. For Western blot applications, polyclonal antibodies like Proteintech's 13534-1-AP perform optimally at dilutions of 1:1000-1:8000, while CUSABIO's polyclonal antibody is recommended at 1:500-1:5000 . For immunohistochemistry, narrower ranges are typically required: 1:50-1:500 for Proteintech antibodies and 1:20-1:200 for CUSABIO's product . Flow cytometry applications generally require less dilution (higher antibody concentration), with some antibodies specifically validated for this purpose. It's important to note that optimal dilutions should be determined empirically for each experimental system, as factors such as expression level, tissue type, and detection method significantly influence performance. Manufacturers often provide starting dilution recommendations based on their validation protocols, but researchers should titrate antibodies in their specific samples to obtain optimal signal-to-noise ratios. Some primary antibodies may also perform differently depending on the secondary detection system employed .

What methods are most effective for detecting P2RX4 expression in different immune cell populations?

Detection of P2RX4 expression in immune cells requires specialized approaches due to variable expression levels across cell types. Gene expression analysis using quantitative RT-PCR represents the most common method for assessing P2RX4 transcripts in isolated immune cells, providing sensitive detection with cell-type specificity . For protein detection, flow cytometry using anti-P2X4 monoclonal antibodies has proven particularly valuable, revealing a distinct hierarchy of expression: highest in eosinophils, followed by neutrophils and monocytes, then basophils and B cells, with lowest expression in T cells . Immunoblotting detects P2RX4 protein in immune cell lysates, though sensitivity may be limited in cells with low expression. Immunohistochemistry and immunofluorescence provide spatial context but require careful antibody validation. For comprehensive characterization, researchers should combine multiple detection methods, as correlation between mRNA and protein levels is not always consistent. When selecting antibodies for immune cell work, those validated specifically in leukocytes should be prioritized, as antibodies validated only in neural tissues may perform differently in immune cells .

How can researchers effectively measure P2RX4 ion channel activity in experimental systems?

Measuring P2RX4 ion channel activity requires specialized techniques that detect either ion flux or channel currents. Ca²⁺ flux assays using cell-permeable calcium-sensitive dyes (Fura-2 or Fluo-4) represent the most widely employed approach, allowing detection of P2RX4 activation through fluorescence microscopy, spectrophotometry, or flow cytometry . The gold standard method, patch-clamp electrophysiology, provides direct measurement of P2RX4 channel currents with high temporal resolution, though it requires specialized equipment and expertise. Alternative approaches include sodium flux assays using sodium green tetra-acetate and dye uptake assays using YO-PRO-1, which enters cells through the dilated pore of activated P2RX4 . When designing these experiments, several considerations are critical: (1) use positive modulators like ivermectin to distinguish P2RX4 from other P2X receptors; (2) employ selective antagonists like 5-BDBD or PSB-12062 as controls; (3) account for potential heteromeric assembly with other P2X subunits, which may alter pharmacological profiles; and (4) validate findings using genetic approaches (siRNA knockdown or CRISPR knockout) to confirm specificity .

What are the recommended protocols for immunohistochemical detection of P2RX4 in different tissue types?

Immunohistochemical detection of P2RX4 requires optimization for specific tissue contexts. For formalin-fixed paraffin-embedded (FFPE) tissues, antigen retrieval is critical—TE buffer at pH 9.0 is most commonly recommended, though citrate buffer at pH 6.0 provides an alternative for certain antibodies and tissues . Blocking of endogenous peroxidases and proteins should precede primary antibody incubation, with optimal dilutions ranging from 1:50-1:500 depending on the specific antibody and tissue. Several P2RX4 antibodies have been validated for specific tissues: Proteintech's 13534-1-AP has been validated in human ovarian cancer tissue, while 66416-1-Ig shows strong reactivity in human kidney tissue . For brain tissue, where P2RX4 demonstrates complex cellular localization patterns, overnight primary antibody incubation at 4°C often yields superior results. Double immunostaining with cell-type markers (such as S100β for astrocytes, Iba1 for microglia, or NeuN for neurons) helps identify specific cell populations expressing P2RX4 . Fluorescent secondary antibodies enable co-localization studies, while chromogenic detection provides better morphological context. Validation should include appropriate negative controls (primary antibody omission and ideally knockout tissue) to confirm staining specificity .

How can P2RX4 antibodies be utilized to investigate heteromeric assembly with other P2X receptor subtypes?

Investigating P2RX4 heteromeric assembly with other P2X receptors requires sophisticated methodological approaches. Co-immunoprecipitation (Co-IP) represents the cornerstone technique, where P2RX4-specific antibodies immunoprecipitate protein complexes that can then be probed for other P2X subunits (particularly P2X6, P2X7 and P2X1) using subtype-specific antibodies . This approach provides direct evidence of physical association, though careful antibody validation is essential to avoid cross-reactivity with related P2X family members. Proximity ligation assays (PLA) offer an alternative in situ approach, allowing visualization of protein interactions at single-molecule resolution in fixed cells or tissues. Förster resonance energy transfer (FRET) between fluorescently tagged P2X subunits provides additional evidence for molecular proximity consistent with heteromeric assembly. Functionally, patch-clamp electrophysiology combined with P2RX4-specific modulators (ivermectin) and inhibitors of other P2X subtypes can reveal pharmacological properties distinct from homomeric channels. For example, P2X1/P2X4 heteromers exhibit slowly desensitizing currents similar to homomeric P2X4 but show significant activation by αβ-MeATP and inhibition by suramin, properties not observed with either subunit alone .

What approaches can researchers use to distinguish between surface-expressed and intracellular pools of P2RX4?

Distinguishing between surface-expressed and intracellular P2RX4 pools requires specialized methodological approaches due to the receptor's significant intracellular localization. Cell surface biotinylation followed by streptavidin pulldown and immunoblotting with P2RX4 antibodies provides quantitative assessment of the plasma membrane fraction. Flow cytometry offers a complementary approach, particularly using non-permeabilized versus permeabilized conditions to differentiate surface from total cellular P2RX4. Fluorescence microscopy with carefully optimized immunostaining protocols can visualize P2RX4 localization, especially when combined with plasma membrane markers (such as wheat germ agglutinin or Na⁺/K⁺-ATPase) and organelle markers (LAMP1 for lysosomes, where P2RX4 is known to accumulate). For higher resolution, confocal or super-resolution microscopy enables precise localization of P2RX4 pools. To study dynamic trafficking, researchers can employ live-cell imaging of fluorescently tagged P2RX4, though careful validation is needed to ensure tagging doesn't disrupt trafficking signals. Functionally, patch-clamp electrophysiology combined with lysosomotropic agents (like vacuolin-1) can help dissect contributions of surface versus lysosomal P2RX4 channels to cellular responses .

How can P2RX4 antibodies be used to investigate the receptor's role in neuroinflammatory conditions?

P2RX4 antibodies serve as crucial tools for investigating the receptor's role in neuroinflammatory conditions, particularly in microglia-mediated pathologies. Immunohistochemical analysis using P2RX4 antibodies can reveal upregulation patterns in activated microglia within inflammatory lesions, as observed in models of neuropathic pain, multiple sclerosis, and ischemic brain injury . Dual immunofluorescence labeling with microglial markers (Iba1, CD11b) and P2RX4 antibodies enables quantification of receptor expression in specific microglial populations and their morphological states. Flow cytometric analysis of isolated CNS immune cells using P2RX4 antibodies provides quantitative assessment of expression levels across multiple cell types simultaneously. For functional studies, P2RX4 antibodies can be used in phosphorylation-specific Western blots to track activation of downstream signaling pathways like PI3K/Akt, which mediates P2RX4-dependent microglial migration and phagocytosis . Co-immunoprecipitation approaches can identify P2RX4 interaction partners that may be differentially regulated during neuroinflammation. In therapeutic contexts, neutralizing antibodies targeting extracellular domains of P2RX4 have been explored as potential treatments for neuroinflammatory conditions. When designing such studies, researchers should include appropriate controls with other purinergic receptors to establish specificity of observed effects to P2RX4 .

What are the most common causes of non-specific binding when using P2RX4 antibodies and how can they be addressed?

Non-specific binding represents a significant challenge when working with P2RX4 antibodies, stemming from several common causes. First, insufficient blocking leads to high background—researchers should optimize blocking protocols using 5% BSA or 5% non-fat milk in TBS-T, potentially adding normal serum from the secondary antibody's host species at 2-5%. Second, excessive primary antibody concentration frequently causes non-specific binding—titration experiments determining optimal dilutions (starting with manufacturer recommendations, typically 1:1000-1:8000 for Western blot and 1:50-1:500 for IHC) are essential . Third, cross-reactivity with related P2X family members may occur, particularly with polyclonal antibodies—validation using P2RX4 knockout/knockdown samples provides the most definitive control. Fourth, tissue fixation artifacts, especially over-fixation, can increase non-specific binding in IHC—optimization of fixation protocols and antigen retrieval methods (comparing citrate buffer pH 6.0 versus TE buffer pH 9.0) is recommended . Finally, secondary antibody cross-reactivity sometimes contributes to background—using highly cross-adsorbed secondary antibodies and including appropriate negative controls (primary antibody omission, isotype controls) helps identify and minimize this issue .

How can researchers address variability in P2RX4 antibody performance across different experimental systems?

Addressing variability in P2RX4 antibody performance requires systematic optimization for each experimental system. First, consider sample preparation variations—different lysis buffers, fixation protocols, or antigen retrieval methods significantly impact epitope accessibility. For Western blotting, compare reducing versus non-reducing conditions, as P2RX4 contains multiple disulfide bonds that may affect antibody recognition . For immunohistochemistry, systematically compare different antigen retrieval methods, as some epitopes may be better exposed with citrate buffer (pH 6.0) while others require TE buffer (pH 9.0) . Second, recognize that P2RX4 undergoes post-translational modifications including glycosylation, which varies across tissues and cell types—this explains the observed molecular weight range of 45-60 kDa rather than the theoretical 43.4 kDa . Third, species-specific differences in P2RX4 sequence may affect antibody binding, particularly for monoclonal antibodies targeting specific epitopes—examine sequence homology in the immunogen region when using antibodies across species. Finally, implement standardized positive controls across experiments (validated P2RX4-expressing cell lines or tissues) to normalize for batch-to-batch antibody variations. When publishing, detailed reporting of antibody catalog numbers, dilutions, and complete protocols facilitates reproducibility .

What approaches can resolve issues with detecting low abundance P2RX4 in certain cell types or tissues?

Detecting low-abundance P2RX4 in challenging samples requires specialized approaches that enhance sensitivity while maintaining specificity. Signal amplification represents the first strategy—tyramide signal amplification (TSA) can increase detection sensitivity by 10-100 fold in immunohistochemistry and immunofluorescence applications. For Western blotting, enhanced chemiluminescence-plus (ECL-plus) reagents and longer exposure times improve detection, while increasing protein loading (up to 50-100 μg per lane) helps when sample quantity isn't limiting . Sample enrichment provides another approach—immunoprecipitation using validated P2RX4 antibodies can concentrate the target protein before detection. For membrane proteins like P2RX4, plasma membrane isolation via differential centrifugation or commercial kits enriches the relevant fraction . For tissues with heterogeneous expression, laser capture microdissection isolates specific P2RX4-expressing cell populations. Alternative detection methods may offer superior sensitivity—droplet digital PCR (ddPCR) at the transcript level or mass spectrometry-based proteomics at the protein level can detect extremely low abundance targets. Finally, consider antibody selection carefully—polyclonal antibodies generally offer higher sensitivity by recognizing multiple epitopes, while high-affinity monoclonal antibodies may provide better signal-to-noise ratios in specific applications .

How might advances in recombinant antibody technology impact future P2RX4 research?

Advances in recombinant antibody technology are poised to transform P2RX4 research through several revolutionary approaches. Single-chain variable fragment (scFv) and nanobody formats derived from conventional P2RX4 antibodies offer superior tissue penetration and reduced immunogenicity for in vivo applications. These smaller antibody fragments can access restricted subcellular compartments where P2RX4 localizes, such as lysosomes and endosomes, enabling more precise tracking of intracellular pools . Creative Biolabs already offers recombinant anti-P2RX4 antibodies that demonstrate high specificity in ELISA applications, representing early adoption of this technology . Future developments will likely include bispecific antibodies simultaneously targeting P2RX4 and other interacting proteins, enabling visualization of protein complexes in situ. Antibody engineering approaches may yield conformation-specific antibodies distinguishing between open/activated and closed/resting states of P2RX4 channels, providing direct readouts of receptor activity. Additionally, recombinant technology facilitates humanization of research antibodies, accelerating translation to clinical applications. The standardization inherent to recombinant production will address current batch-to-batch variability issues, enhancing reproducibility across laboratories and experimental systems .

What emerging methodologies might enhance the specificity and utility of P2RX4 antibodies in complex tissue environments?

Emerging methodologies promise to revolutionize P2RX4 detection specificity in complex tissues through several innovative approaches. Proximity labeling techniques, including APEX2 and BioID fused to P2RX4, are enabling mapping of the receptor's proximal interactome in specific cellular compartments, providing functional context beyond mere localization . Mass cytometry (CyTOF) using metal-conjugated P2RX4 antibodies allows simultaneous analysis of P2RX4 expression alongside dozens of other markers in heterogeneous tissue samples, revealing previously unappreciated cell-type specific expression patterns. Spatial transcriptomics approaches, when combined with P2RX4 immunohistochemistry, correlate protein localization with transcriptional signatures of surrounding cells, providing insights into local microenvironmental influences on P2RX4 function . Expansion microscopy physically enlarges specimens, separating densely packed epitopes and enhancing resolution of P2RX4 distribution in complex tissues like brain. Additionally, CRISPR-based epitope tagging of endogenous P2RX4 avoids artifacts associated with antibody cross-reactivity and overexpression systems. For therapeutic applications, antibody-drug conjugates targeting cell-surface P2RX4 in pathological conditions like neuropathic pain or neuroinflammation represent an exciting frontier, potentially offering precise modulation of P2RX4 signaling in specific cell populations .

How can computational approaches improve P2RX4 antibody design and epitope targeting?

Computational approaches are transforming P2RX4 antibody development through multiple sophisticated strategies. Structure-based epitope prediction, leveraging high-resolution structures of P2RX4 (including recent cryo-EM data), now enables rational targeting of functionally crucial regions such as ATP-binding domains or conformationally distinct elements involved in channel gating. These computationally identified epitopes can generate antibodies that specifically modulate receptor function rather than merely detecting protein presence . Machine learning algorithms analyzing large datasets of antibody-antigen interactions are improving prediction of epitope accessibility in native versus denatured states, guiding development of application-specific antibodies optimized for either Western blotting or immunohistochemistry/cytometry . Molecular dynamics simulations reveal transient conformations of P2RX4, identifying previously unrecognized epitopes that may distinguish receptor activation states. For antibodies targeting cross-species applications, sequence conservation analysis across mammalian P2RX4 orthologs identifies evolutionarily constrained epitopes likely to enable broad reactivity profiles. Additionally, immunogenicity prediction algorithms help design antibodies with reduced risk of generating neutralizing responses in therapeutic applications. These computational approaches not only enhance antibody specificity and functionality but also accelerate development timelines by reducing empirical screening requirements and focusing wet-lab validation efforts on the most promising candidates .