xpr1 Antibody

What is XPR1 and why is it important in research?

XPR1 (Xenotropic and Polytropic Retrovirus Receptor 1) is an 8-pass transmembrane molecule consisting of 696 amino acids that was initially identified as a cell surface receptor for xenotropic and polytropic murine leukemia retroviruses (X- and P-MLV) . Recent research has revealed that XPR1 functions as a phosphate exporter leading to depletion of intracellular phosphate and regulates polyphosphate (polyP) in platelets . XPR1 is critical for studying:

• Phosphate homeostasis mechanisms across different cell types

• Thrombosis and platelet function

• Neurological disorders, particularly primary familial brain calcification (PFBC)

• Retroviral infection mechanisms

• Cell signaling pathways involving G-protein-coupled receptors

XPR1 contains an SPX domain in its amino-terminal region that is also found in yeast and plant proteins involved in phosphate homeostasis . This evolutionary conservation highlights its fundamental importance in cellular function.

What types of XPR1 antibodies are available for research applications?

XPR1 antibodies are primarily available as rabbit polyclonal antibodies that target different epitopes of the protein. Most commercial XPR1 antibodies are:

These antibodies typically recognize XPR1 with an observed molecular weight of approximately 82-84 kDa in Western blot applications .

What are the validated applications for XPR1 antibodies?

XPR1 antibodies have been validated for multiple research applications:

• Western Blot (WB): Detecting XPR1 protein in cell/tissue lysates (dilutions typically 1:500-1:5000)

• Immunohistochemistry (IHC): Localizing XPR1 in tissue sections (dilutions typically 1:20-1:200)

• Immunofluorescence (IF/ICC): Visualizing subcellular localization

• Flow Cytometry (FC): Analyzing XPR1 expression in cell populations

• ELISA: Quantitative detection of XPR1 in samples

Research shows successful detection of XPR1 in various samples, including:

• Human and murine platelet membrane fractions showing a single band at ~65 kDa

• Jurkat cells for WB and flow cytometry applications

• Human heart tissue for IHC applications

• HEK293 cells and MEG-01 megakaryocytes in XPR1 overexpression studies

How should XPR1 antibodies be validated for specificity in experimental designs?

When validating XPR1 antibodies for research, consider the following methodological approaches:

• Knockdown/Knockout Controls: Use siRNA-mediated XPR1 knockdown samples as negative controls to confirm antibody specificity . This approach has been used successfully to validate both the role of XPR1 in phosphate efflux and antibody specificity.

• Overexpression Systems: Compare signal intensity between mock-transfected cells and XPR1-overexpressing cells. Studies have shown 8 to 45-fold increases in XPR1 signal intensity in Western blots of transfected cells compared to controls .

• Recombinant Protein Controls: Use recombinant human XPR1 expressed in HEK293 cells as a positive control for antibody validation .

• Cross-Species Reactivity Tests: Test antibody performance across human, mouse, and rat samples to confirm conservation of the epitope. Many XPR1 antibodies show cross-reactivity with multiple species .

• Subcellular Fractionation: Since XPR1 is a membrane protein, validation should include membrane fraction enrichment protocols to confirm localization .

What are the optimal sample preparation methods for detecting XPR1 in different applications?

Sample preparation is critical for successful XPR1 detection:

For Western Blot:

• Prepare membrane fractions for optimal XPR1 detection, as it is a transmembrane protein

• Use SDS-PAGE with standard protocols, detecting XPR1 at approximately 82-84 kDa

• Note that XPR1 may show abnormal migration patterns, with some researchers reporting bands between 65-100 kDa

For Immunohistochemistry:

• Use paraffin-embedded tissues with antigen retrieval

• Dilute primary antibodies between 1:20-1:200 depending on the antibody

• Human heart tissue has been successfully used for XPR1 IHC validation

For Flow Cytometry:

• Fix cells with 90% methanol

• Block with 3% BSA for 30 minutes

• Use approximately 0.25 μg antibody per 10^6 cells in a 100 μl suspension

• Jurkat cells have been successfully used for flow cytometry applications

What are the common troubleshooting issues when working with XPR1 antibodies?

Researchers should be aware of these common challenges:

• Variable Molecular Weight Detection: XPR1 may appear at different molecular weights (65-100 kDa) depending on the cell type and post-translational modifications . This is not necessarily indicative of antibody non-specificity.

• Membrane Protein Extraction Challenges: As an 8-pass transmembrane protein, XPR1 may be difficult to extract completely. Use specialized membrane protein extraction buffers containing appropriate detergents.

• Species-Specific Differences: When working across species, be aware that although the protein is conserved, there may be epitope differences that affect antibody recognition.

• Background Signal in IHC/IF: Optimize blocking conditions (3% BSA has been reported as effective) and antibody dilutions to minimize background .

• Storage Stability Issues: Store antibodies at -20°C and avoid repeated freeze-thaw cycles to maintain activity . Follow manufacturer recommendations for storage conditions.

How can XPR1 antibodies be utilized to study phosphate transport mechanisms?

XPR1 antibodies can be powerful tools for investigating phosphate homeostasis:

• Phosphate Efflux Assays: Combine XPR1 immunodetection with functional studies of phosphate export. This approach has revealed that XPR1 overexpression reduces total polyP content to 58-65% compared to control cells, while siRNA-mediated knockdown of XPR1 increases polyP levels .

• Mutation Impact Studies: XPR1 antibodies can detect mutant forms of the protein to correlate expression levels with functional changes. For example, antibodies have been used to confirm plasma membrane localization of XPR1 variants associated with PFBC while studying their impact on phosphate efflux .

• Co-immunoprecipitation Studies: Use XPR1 antibodies to identify interacting partners in phosphate transport pathways.

• Subcellular Localization: Track XPR1 trafficking in response to phosphate availability using immunofluorescence approaches.

• Mechanistic Studies: Investigate how XPR1 coordinates with other phosphate transporters like PiT1 and PiT2 through immunodetection of all three transporters simultaneously .

What approaches can be used to study the role of XPR1 in thrombosis and platelet function?

Recent research has identified XPR1 as a regulator of polyphosphate in platelets with implications for thrombosis . Consider these methodological approaches:

• Platelet Isolation Protocols: Carefully prepare platelet membrane fractions for XPR1 detection using antibodies against both N and C termini, which have successfully detected a single band at ~65 kDa in human and mouse platelets .

• Thrombin Generation Assays: Combine XPR1 antibody-based detection with real-time thrombin generation assays in platelet-rich plasma to correlate XPR1 expression with platelet reactivity .

• Platelet Activation Studies: Use flow cytometry with XPR1 antibodies to study expression changes following platelet activation.

• In Vivo Thrombosis Models: Apply XPR1 antibodies for immunohistochemical detection in tissue samples from thrombosis models.

• Correlation Studies: Investigate the relationship between XPR1 expression levels (detected via antibodies) and polyP-mediated procoagulant activities that lead to enhanced clotting in human plasma .

How can researchers investigate XPR1's role in primary familial brain calcification (PFBC)?

XPR1 mutations have been implicated in PFBC, a rare neurodegenerative disorder characterized by calcium phosphate deposits in the basal ganglia . Methodological approaches include:

• Mutation Screening and Functional Correlation: Use XPR1 antibodies to detect protein expression levels of different variants identified in PFBC patients (e.g., p.Leu145Pro, p.Ser136Asn, p.Leu140Pro, and p.Leu218Ser) .

• Phosphate Efflux Complementation Assays: Combine XPR1 knockdown with re-expression of wild-type or mutated XPR1, using antibodies to confirm expression while measuring functional outputs .

• Brain Tissue Analysis: Apply IHC with XPR1 antibodies to study expression patterns in brain regions affected by calcification.

• Comparative Studies: Investigate relationships between XPR1, SLC20A2, PDGFB, and PDGFRB (all genes associated with PFBC) using respective antibodies for co-localization or expression level comparisons .

• Plasma Membrane Localization: Confirm proper localization of wild-type and mutant XPR1 using immunofluorescence techniques .

What considerations are important when using XPR1 antibodies to study its retroviral receptor function?

XPR1 was initially identified as a receptor for xenotropic and polytropic murine leukemia retroviruses . To study this aspect:

• Infection Assays: Use XPR1 antibodies to correlate expression levels with cellular susceptibility to X-MLV and P-MLV infection .

• Blocking Studies: Test whether pre-incubation with certain XPR1 antibodies can block viral entry.

• Structure-Function Analysis: Study both wild-type and mutant XPR1 forms using antibodies to understand which domains are critical for viral binding versus phosphate transport. For example, research has shown that the p.Leu145Pro-mutated XPR1 neither re-establishes phosphate efflux nor serves as a receptor for X-MLV infection .

• Signal Transduction Studies: Investigate how retroviral binding to XPR1 disrupts cAMP-mediated signaling leading to apoptosis .

• Comparative Expression Analysis: Use antibodies to quantify XPR1 levels across different cell types and correlate with viral susceptibility.

How should researchers interpret contradictory data between XPR1 protein expression and function?

When facing contradictions between XPR1 expression and functional data:

• Protein Modification Analysis: Consider that post-translational modifications may affect function without changing expression levels detectable by antibodies.

• Subcellular Localization Discrepancies: Verify whether XPR1 is properly localized to the plasma membrane despite normal expression levels. Some PFBC-associated mutations show normal expression but impaired function due to trafficking defects .

• Splice Variant Considerations: Use antibodies targeting different epitopes to determine if alternative splice variants might explain functional differences.

• Binding Partner Alterations: Consider that XPR1 may require specific interacting proteins for its function that are not present in all experimental systems.

• Quantification Methods: Compare relative vs. absolute quantification methods when measuring XPR1 levels in experimental systems.

What experimental design approaches can address the multifunctional nature of XPR1?

XPR1 functions as both a phosphate exporter and retroviral receptor, requiring sophisticated experimental designs:

• Domain-Specific Mutation Analysis: Use XPR1 antibodies to confirm expression of domain-specific mutants while testing for selective loss of function (phosphate export vs. viral receptor activity) .

• Complementation Studies: Design rescue experiments with structurally distinct XPR1 homologs (such as yeast Pho91 or T. cruzi TcPho91) and use species-specific antibodies to confirm expression .

• Temporal Analysis: Study the dynamics of XPR1 function using inducible expression systems with antibody-based monitoring of protein levels over time.

• Contextual Dependencies: Investigate XPR1 function across different cell types where phosphate homeostasis requirements may vary.

• Cross-Disciplinary Approaches: Combine techniques from virology, cell signaling, and mineral homeostasis fields to comprehensively understand XPR1 functionality.

How can researchers address specificity concerns when studying XPR1 in complex biological samples?

To ensure specificity when detecting XPR1 in complex samples:

• Multiple Antibody Validation: Use antibodies targeting different epitopes of XPR1 to confirm findings, as demonstrated in studies using antibodies against both N and C termini .

• Genetic Controls: Include XPR1 knockout/knockdown samples as negative controls and XPR1-overexpressing samples as positive controls .

• Tissue-Specific Expression Profiles: Create a reference database of XPR1 expression across tissues using validated antibodies to identify regions of high and low expression.

• Cross-Reactivity Analysis: Test antibodies against closely related proteins or in heterologous expression systems to confirm specificity.

• Mass Spectrometry Validation: Confirm the identity of immunoprecipitated proteins using mass spectrometry to validate antibody specificity in pull-down experiments.

How might XPR1 antibodies be utilized in diagnostic development for PFBC and related disorders?

XPR1 mutations account for approximately 5.5% of PFBC cases in French cohorts and 2.5% in North American cohorts . Research opportunities include:

• Biomarker Development: Investigate whether XPR1 protein levels or phosphorylation states in accessible biofluids correlate with disease progression.

• Patient Stratification: Utilize XPR1 antibodies in immunohistochemical analyses of patient samples to determine if expression patterns correlate with specific clinical presentations.

• Functional Diagnostics: Develop cell-based assays using XPR1 antibodies to assess the pathogenicity of novel variants of uncertain significance.

• Cross-Comparison Studies: Design methodologies to distinguish between PFBC caused by XPR1 mutations versus those caused by SLC20A2, PDGFB, or PDGFRB mutations through protein expression analyses .

• Blood-Based Testing: Explore peripheral blood cell expression of XPR1 as a potential surrogate marker for cerebral phosphate homeostasis disturbances.

What methodological approaches can advance understanding of XPR1's role in cell signaling beyond phosphate transport?

XPR1 has been identified as an atypical G-protein-coupled receptor , suggesting broader signaling functions:

• Signalosome Analysis: Use XPR1 antibodies in proximity ligation assays to identify proteins interacting with XPR1 in different cellular contexts.

• Phosphorylation Studies: Develop phospho-specific XPR1 antibodies to track activation states of the protein in response to various stimuli.

• Real-Time Monitoring: Combine XPR1 antibodies with live-cell imaging techniques to visualize dynamic changes in localization during signaling events.

• Single-Cell Analysis: Apply XPR1 antibodies in single-cell analysis workflows to understand cell-to-cell variability in expression and function.

• Conditional Knockout Models: Develop tissue-specific XPR1 knockout models and use antibodies to confirm deletion while studying downstream signaling consequences.