CXE17 Antibody

What is CXCR7 and why is it an important research target?

CXCR7 (C-X-C chemokine receptor type 7), also known as RDC1, is a member of the G-protein coupled receptor family. It functions as a receptor that can bind chemokines CXCL11 and CXCL12 with high affinity and also acts as a coreceptor with other signaling molecules. CXCR7 plays crucial roles in multiple biological processes including cell migration, adhesion, and survival in both normal and pathological conditions .

Research on CXCR7 is particularly important because:

• It participates in immune cell chemotaxis and chemokine-mediated signaling

• It's involved in cellular responses to interferons

• It contributes to regulation of leukocyte differentiation

• It has significant implications in disease processes including immune disorders and cancer development

What are the most common applications for CXCR7 antibodies in laboratory research?

CXCR7 antibodies are versatile research tools with multiple validated applications:

These applications have been validated in multiple published studies, with Western blot applications appearing in at least 14 publications, IHC in 8 publications, and IF in 3 publications according to available data .

What cell and tissue types are commonly used for CXCR7 expression studies?

CXCR7 antibodies have been validated in various cell and tissue types:

For Western blot applications:

• Human cell lines: Raji cells, Jurkat cells, K-562 cells

• Mouse tissues: thymus tissue, spleen tissue

For immunohistochemistry:

• Mouse brain tissue (with recommended antigen retrieval using TE buffer pH 9.0 or citrate buffer pH 6.0)

For immunoprecipitation:

• HUVEC cells (Human Umbilical Vein Endothelial Cells)

For flow cytometry:

• Neural progenitor cells

Selection of appropriate cell or tissue types should be based on the specific research question and known expression patterns of CXCR7 in different biological contexts.

How should researchers optimize CXCR7 antibody dilutions for different applications?

Optimizing antibody dilutions is crucial for obtaining specific signals while minimizing background. For CXCR7 antibodies:

• Begin with manufacturer-recommended dilutions:

  • Western Blot: Start with 1:500-1:1000 dilution

  • Immunohistochemistry: Begin with 1:50-1:500 range

  • Immunoprecipitation: Use 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate

• Perform titration experiments:

  • Test 3-4 dilutions within and beyond the recommended range

  • Include appropriate positive and negative controls

  • Evaluate signal-to-noise ratio at each dilution

• Consider sample-specific optimizations:

  • Different cell lines or tissues may require adjusted dilutions

  • Check validation data for your specific sample type

• Document optimization parameters:

  • Record blocking conditions, incubation times/temperatures, and detection methods

  • Note lot-to-lot variations that may necessitate re-optimization

Remember that optimal dilutions are application, sample, and laboratory-specific. The recommendation to "titrate this reagent in each testing system to obtain optimal results" emphasizes the importance of empirical determination for each experimental setup .

What are the best practices for antigen retrieval when using CXCR7 antibodies in immunohistochemistry?

Effective antigen retrieval is critical for successful immunohistochemical detection of CXCR7. Based on validated protocols:

• Primary recommended method:

  • Use TE buffer at pH 9.0

  • Heat-induced epitope retrieval (microwave or pressure cooker)

  • Monitor temperature and time carefully for reproducibility

• Alternative method:

  • Citrate buffer at pH 6.0

  • Heat-induced epitope retrieval

  • May be preferred for certain tissue types or fixation conditions

• Method optimization considerations:

  • Fixation type and duration influence retrieval efficiency

  • Fresh frozen vs. formalin-fixed paraffin-embedded tissues require different approaches

  • Pilot studies comparing multiple retrieval methods are recommended for new tissue types

• Controls for antigen retrieval:

  • Include known positive tissue controls (e.g., mouse brain tissue for CXCR7)

  • Process serial sections with and without retrieval to assess enhancement

  • Monitor tissue morphology to avoid over-retrieval damage

The optimal retrieval method may vary depending on tissue source, fixation protocol, and specific antibody clone used. Systematic optimization is essential for reliable and reproducible IHC results.

How can researchers confirm the specificity of CXCR7 antibody staining?

Confirming antibody specificity is fundamental to reliable research outcomes. For CXCR7 antibodies, employ multiple validation approaches:

• Positive and negative controls:

  • Use cell lines or tissues with known CXCR7 expression (e.g., Raji cells, Jurkat cells)

  • Include negative controls: isotype controls, secondary antibody-only controls

  • Consider using CXCR7 knockout/knockdown samples as definitive negative controls

• Multiple detection methods:

  • Confirm findings using different techniques (WB, IHC, IF, flow cytometry)

  • Compare results across different antibody clones targeting distinct epitopes

  • Correlate protein detection with mRNA expression data where possible

• Peptide competition assays:

  • Pre-incubate antibody with immunizing peptide before application

  • Specific signal should be significantly reduced or eliminated

  • Non-specific binding will typically remain unchanged

• Molecular weight verification:

  • In Western blots, confirm detection at the expected molecular weight (CXCR7: 45-50 kDa observed vs. 41 kDa calculated)

  • Assess any additional bands for biological relevance (modified forms, degradation products)

• Subcellular localization assessment:

  • Compare observed localization with expected patterns (e.g., membrane localization for CXCR7)

  • Use co-localization with known markers to confirm appropriate distribution

Published studies using knockout/knockdown validation provide strong evidence for antibody specificity, with at least 4 publications documenting such validation for CXCR7 antibodies .

How can CXCR7 antibodies be used to study receptor dynamics in response to chemokine stimulation?

CXCR7 antibodies enable investigation of receptor trafficking and dynamics following chemokine stimulation:

• Receptor polarization studies:

  • Examine CXCR7 redistribution in response to CXCL12 gradients

  • Use immunofluorescence to visualize receptor clustering and polarization

  • Compare with other chemokine receptors (e.g., CXCR4) to assess differential responses

• Internalization assays:

  • Track surface-to-intracellular movement following ligand binding

  • Use flow cytometry to quantify surface expression changes over time

  • Combine with endocytic pathway markers to define trafficking routes

• Live-cell imaging approaches:

  • Use fluorescently-tagged antibodies for real-time receptor tracking

  • Create stable gradients of chemokines to observe directional responses

  • Quantify receptor movement kinetics and cytoskeletal reorganization

• Analyzing multi-receptor interactions:

  • Study CXCR7 co-localization with CXCR4 after CXCL12 stimulation

  • Examine cytoskeletal rearrangements (F-actin) in relation to receptor redistribution

  • Evaluate the spatial relationship between receptors during cell polarization events

As demonstrated in HUVECs, CXCL12 stimulation induces polarization of both CXCR4 and CXCR7, along with F-actin reorganization, suggesting coordinated receptor dynamics during chemokine-induced cell polarization .

What are the methodological considerations for analyzing CXCR7 in complex transcriptional profiling studies?

Integrating CXCR7 analysis into transcriptional profiling requires sophisticated methodological approaches:

• Single-cell RNA sequencing (scRNA-seq) approaches:

  • Identify cell populations expressing CXCR7

  • Track changes in CXCR7 expression across different experimental conditions

  • Integrate with other chemokine receptor and signaling pathway components

• Differential expression analysis:

  • Compare CXCR7 expression between experimental and control groups

  • Apply appropriate statistical thresholds (e.g., two-fold change)

  • Integrate with pseudobulk RNA-seq for aggregated cell population analysis

• Pathway and network analysis:

  • Construct protein-protein interaction networks including CXCR7

  • Use tools like STRING for network visualization

  • Perform community detection to identify functional clusters

  • Apply Gene Ontology enrichment and Gene Set Enrichment Analysis

• Validation of transcriptional findings:

  • Correlate RNA expression with protein detection using CXCR7 antibodies

  • Perform functional studies to confirm biological relevance of expression changes

  • Consider temporal dynamics of expression changes in longitudinal studies

In advanced studies, researchers have identified that CXCR7 and related pathway genes show altered expression in disease models, with changes in pathways related to immune cell chemotaxis, chemokine-mediated signaling, and cellular responses to interferons being particularly significant .

How can CXCR7 antibodies be used in therapeutic development and mechanistic studies?

CXCR7 antibodies serve as valuable tools in therapeutic research contexts:

• Target validation studies:

  • Use antibodies to confirm CXCR7 presence in disease-relevant tissues

  • Correlate receptor expression with disease progression markers

  • Assess changes in receptor expression following experimental interventions

• Mechanism of action studies:

  • Track downstream signaling pathways affected by receptor modulation

  • Perform co-immunoprecipitation to identify interaction partners

  • Evaluate effects of receptor neutralization on cellular phenotypes

• Therapeutic antibody development:

  • Use research-grade antibodies as references for therapeutic antibody development

  • Compare binding characteristics and epitope specificity

  • Assess functional effects across different antibody clones

• Preclinical model development:

  • Apply antibodies in disease models to evaluate receptor involvement

  • Track treatment responses at transcriptional and protein levels

  • Use RNA-seq to identify off-target effects and safety signals

Research has demonstrated that antibodies targeting the CXCL12-CXCR7 axis can modulate immune cell behavior, affecting pathways related to cellular response to interferons and lymphocyte chemotaxis, with potential therapeutic implications in immune-mediated diseases .

What are common challenges in Western blot detection of CXCR7 and how can they be addressed?

Western blot detection of CXCR7 presents several technical challenges:

• Molecular weight discrepancies:

  • Calculated molecular weight (41 kDa) vs. observed weight (45-50 kDa)

  • Solution: Consider post-translational modifications (glycosylation, phosphorylation)

  • Include positive control lysates (e.g., Raji cells, mouse thymus tissue)

• Membrane protein extraction issues:

  • CXCR7 is a transmembrane protein that may be difficult to extract

  • Solution: Use specialized membrane protein extraction buffers containing appropriate detergents

  • Avoid excessive heating which can cause protein aggregation

• Non-specific binding:

  • Multiple bands or high background may appear

  • Solution: Optimize blocking conditions (5% BSA often better than milk for phospho-proteins)

  • Increase washing duration and frequency

  • Consider using reducing conditions as specified in protocols

• Sample preparation considerations:

  • Fresh vs. frozen tissue influences protein integrity

  • Solution: Standardize sample collection and storage protocols

  • Add protease and phosphatase inhibitors immediately during lysis

  • Process samples consistently to minimize variation

For optimal CXCR7 detection, Western blot experiments should be conducted under reducing conditions using immunoblot buffer group 8 as validated in published protocols .

How can researchers optimize flow cytometry protocols for CXCR7 detection?

Flow cytometry optimization for CXCR7 requires attention to several methodological details:

• Cell preparation considerations:

  • Single-cell suspensions must be prepared without damaging surface receptors

  • Avoid harsh enzymatic dissociation methods that might cleave CXCR7

  • Keep cells at appropriate temperature to prevent receptor internalization

• Antibody selection and titration:

  • Choose antibodies validated for flow cytometry applications

  • Test multiple concentrations to determine optimal signal-to-noise ratio

  • Include appropriate isotype controls at matching concentrations

• Staining protocol optimization:

  • Evaluate different staining buffers (PBS/BSA vs. commercial buffers)

  • Test various incubation times and temperatures

  • For indirect staining, select compatible secondary antibodies (e.g., NorthernLights™ 557-conjugated anti-sheep IgG)

• Multiparameter panel design:

  • Choose fluorophores with minimal spectral overlap

  • Include viability dye to exclude dead cells

  • Consider co-staining with CXCR4 to examine receptor co-expression patterns

• Gating strategy development:

  • Use FMO (fluorescence minus one) controls for boundary setting

  • Compare expression between known positive populations (e.g., neural progenitor cells) and negative controls

  • Gate on singlets and viable cells before analyzing receptor expression

Successful flow cytometry detection of CXCR7 has been demonstrated in neural progenitor cells using specific staining protocols that can serve as a starting point for optimization in other cell types .

What factors affect reproducibility in CXCR7 antibody-based experiments and how can they be controlled?

Ensuring reproducibility in CXCR7 antibody experiments requires systematic control of multiple variables:

• Antibody-related factors:

  • Lot-to-lot variation can significantly impact results

  • Solution: Document lot numbers and validate each new lot

  • Consider creating large aliquots of validated lots for long-term studies

  • Store antibodies according to manufacturer specifications (-20°C, avoid freeze-thaw cycles)

• Sample preparation consistency:

  • Standardize collection, fixation, and processing protocols

  • Document and control fixation duration and conditions

  • For tissues, use consistent sectioning techniques and thickness

  • For cells, maintain consistent passage numbers and confluency

• Technical execution variables:

  • Develop detailed standard operating procedures (SOPs)

  • Control for timing of each experimental step

  • Standardize equipment settings and calibration

  • Implement quality control checkpoints throughout protocols

• Data acquisition and analysis:

  • Use consistent acquisition parameters (exposure times, gain settings)

  • Apply standardized analysis pipelines and gating strategies

  • Document software versions and analysis parameters

  • Consider blinded analysis to reduce unconscious bias

• Biological variability management:

  • Account for sex, age, and genetic background in animal studies

  • Control for cell density and passage number in cell culture

  • Consider circadian or cyclical variations in protein expression

  • Include sufficient biological replicates (minimum n=3, preferably more)

Implementing these controls can significantly improve reproducibility across experiments, laboratories, and publication records for CXCR7 research.

How are CXCR7 antibodies being used to understand intercellular communication in complex tissue environments?

Antibodies against CXCR7 are enabling new insights into intercellular communication:

• Spatial transcriptomics integration:

  • Correlate CXCR7 protein localization with spatial gene expression patterns

  • Map receptor distribution across tissue niches and microenvironments

  • Identify cellular interactions through complementary receptor-ligand expression

• Multi-receptor signaling network analysis:

  • Study how CXCR7 functions alongside CXCR4 in response to CXCL12

  • Investigate competitive or cooperative binding between receptors

  • Analyze downstream pathway activation through differential receptor engagement

• Tissue-specific communication circuits:

  • Examine CXCR7 expression in specialized tissues (brain, immune organs)

  • Map chemokine gradients in relation to receptor expression

  • Analyze functional outcomes of blocking specific receptor-ligand interactions

• Cell polarization and directional migration:

  • Use antibodies to track receptor redistribution during polarization

  • Correlate receptor clustering with cytoskeletal reorganization

  • Analyze the temporal sequence of molecular events during chemotactic responses

Research using CXCR7 antibodies has revealed that receptor polarization occurs rapidly (within 5 minutes) following CXCL12 stimulation in HUVECs, coordinating with cytoskeletal changes to facilitate directional cell responses .

What are the latest approaches for using CXCR7 antibodies in immunotherapy research?

CXCR7 antibodies are finding increased applications in immunotherapy research:

• Immune cell modulation strategies:

  • Track CXCR7 expression on different immune cell populations

  • Assess how receptor blockade affects immune cell trafficking and function

  • Investigate the impact on inflammatory responses and disease progression

• Transcriptional profiling of therapeutic responses:

  • Use RNA-seq to analyze gene expression changes following antibody treatment

  • Identify key regulated pathways (e.g., type I and II interferon responses)

  • Cluster differentially expressed genes into functional networks

• Off-target effect assessment:

  • Perform comprehensive analysis of antibody-specific DEGs

  • Evaluate biological processes affected by antibody treatment beyond target pathways

  • Assess safety profiles by examining minimal off-target signatures

• Combination therapy approaches:

  • Test CXCR7-targeting antibodies with other immune modulators

  • Analyze synergistic effects on immune cell polarization and function

  • Evaluate pathway-specific versus broad immunomodulatory effects

Recent research shows that antibodies targeting the CXCL12-CXCR7 axis demonstrate relatively specific effects, with minimal off-target pathway modulation compared to disease-related changes, suggesting favorable safety profiles for therapeutic development .

How can researchers leverage antibody sequence data mining for improved CXCR7 antibody development?

Advanced data mining approaches offer new opportunities for antibody development:

• Sequence database utilization:

  • Mine extensive collections of antibody sequences for structural insights

  • Identify conserved regions with optimal target binding characteristics

  • Apply database searching in publicly available proteomics data

• Cross-reactivity prediction:

  • Analyze sequence similarities between human and model organism CXCR7

  • Predict epitope-specific binding across species

  • Design antibodies with controlled cross-reactivity profiles

• Epitope mapping enhancements:

  • Use sequence data to predict optimal epitope targets

  • Design antibodies targeting specific receptor domains (N-terminal, extracellular loops)

  • Compare functional outcomes of different epitope-targeting approaches

• Therapeutic antibody optimization:

  • Analyze sequences of research-grade antibodies with desirable properties

  • Modify sequences to enhance specificity, affinity, or functional properties

  • Apply computational modeling to predict binding characteristics of novel designs

This emerging field connects basic research antibodies with therapeutic development pipelines, leveraging increasing availability of sequence databases and computational tools to accelerate antibody engineering and optimization .