CXE7 Antibody

What are the primary applications for CXCR7 antibodies in research?

CXCR7 antibodies are versatile tools applicable across multiple experimental techniques. The most common applications include Western Blot (WB), Immunohistochemistry (IHC), Immunofluorescence (IF), Immunoprecipitation (IP), and ELISA. Each application requires specific optimization parameters for optimal results. For Western Blotting, a dilution range of 1:500-1:1000 is typically recommended, while IHC applications usually require dilutions between 1:50-1:500 . For immunoprecipitation, 0.5-4.0 μg of antibody is typically used for 1.0-3.0 mg of total protein lysate . These parameters should be titrated in each experimental system to achieve optimal signal-to-noise ratios.

What is the species reactivity profile of CXCR7 antibodies?

Most commercially available CXCR7 antibodies demonstrate reactivity with human, mouse, and rat samples . This cross-species reactivity is particularly valuable for translational research that bridges findings between animal models and human tissues. When selecting an antibody for your research, it's important to verify the validated reactivity profile, as some antibodies may show differential sensitivity across species. Published literature has confirmed the reactivity of certain CXCR7 antibodies across multiple model systems, making them suitable for comparative studies across species .

What is the expected molecular weight of CXCR7 in Western blot applications?

While the calculated molecular weight of CXCR7 is approximately 41 kDa (based on 362 amino acids), the observed molecular weight in experimental settings typically ranges between 45-50 kDa . This discrepancy is primarily attributed to post-translational modifications, particularly glycosylation of the receptor. When interpreting Western blot results, researchers should expect bands within this range rather than precisely at the calculated molecular weight. Validation data from multiple cell lines including Raji, Jurkat, and K-562 cells consistently show bands in this range .

What are the common technical challenges when detecting CXCR7 in tissue samples?

Detection of CXCR7 in tissue samples can present several technical challenges. For IHC applications in brain tissue, antigen retrieval is typically required. The recommended protocol involves TE buffer at pH 9.0, although citrate buffer at pH 6.0 may serve as an alternative . Signal specificity can be another challenge, particularly in tissues with low expression levels. To overcome this, researchers should:

• Optimize antibody concentration through systematic titration

• Extend primary antibody incubation times (potentially overnight at 4°C)

• Employ signal amplification systems for low-abundance targets

• Include appropriate positive controls (e.g., Raji cells, Jurkat cells for Western blot)

• Validate specificity through knockdown/knockout controls

How can I validate the specificity of CXCR7 antibody staining?

Validating antibody specificity is crucial for reliable research outcomes. Multiple approaches should be employed:

• Knockout/Knockdown controls: Compare staining between wild-type and CXCR7-deficient samples. Several publications have utilized this approach for validation .

• Peptide competition assays: Pre-incubation of the antibody with the immunizing peptide should abolish specific staining.

• Multiple antibody validation: Use antibodies from different sources targeting distinct epitopes to confirm staining patterns.

• Correlation with mRNA expression: Combine protein detection with mRNA analysis (e.g., RT-PCR, RNAscope) to confirm expression patterns.

• Positive and negative tissue controls: Include tissues with known high expression (e.g., certain cancer tissues) and low/no expression as controls.

How does CXCR7 expression correlate with cancer progression and metastasis?

CXCR7 has been linked to tumor growth, survival, and metastasis across multiple aggressive cancer types, including breast cancer, glioblastoma, and prostate cancer . Research indicates that CXCR7 overexpression correlates with enhanced tumor angiogenesis and immune evasion mechanisms. When investigating CXCR7 in cancer models, researchers should consider:

• Differential expression patterns between primary tumors and metastatic lesions

• Co-expression with CXCR4, as these receptors often function cooperatively

• Correlation between CXCR7 expression and clinical outcomes

• Changes in expression during treatment response and resistance development

Blocking CXCR7-mediated pathways has shown promising results in suppressing tumor progression, highlighting this receptor as a potential therapeutic target .

What methodologies are recommended for detecting CXCR7 in cancer tissue samples?

Detection of CXCR7 in cancer tissues requires specific methodological considerations. For immunohistochemical analysis of breast cancer tissue, successful protocols have employed a working concentration of 0.5 μg/mL with a 1-hour room temperature incubation . Heat-induced epitope retrieval using basic antigen retrieval reagents is typically required before primary antibody application.

For fluorescence detection in cancer cell lines such as MCF-7 (breast cancer), protocols utilizing 8 μg/mL antibody concentration with 3-hour room temperature incubation have yielded specific staining localized to both cell surface and cytoplasm . This dual localization is consistent with the receptor's known trafficking between membrane and intracellular compartments.

When analyzing cancer tissues:

• Include both tumor and adjacent normal tissue for comparison

• Assess expression in different cellular compartments (membrane vs. cytoplasm)

• Quantify staining intensity using digital image analysis when possible

• Correlate expression with tumor grade, stage, and molecular subtypes

How can CXCR7 antibodies be utilized in single-cell analysis platforms?

Advanced single-cell analysis platforms, including those utilizing antibody capture technologies like those provided by 10x Genomics, can incorporate CXCR7 antibodies for protein detection at the single-cell level. When designing such experiments:

• Ensure antibodies are compatible with the specific platform's conjugation chemistry

• Validate antibody performance in the context of the multiplexed panel

• Include appropriate isotype controls to assess background binding

• Analyze data using specialized software that can visualize antibody-derived counts alongside transcriptomic data

For analysis of antibody capture data, log-transformed antibody counts are typically used for dimensionality reduction analyses, which differs from the approach used for gene expression data where PCA-reduced space from raw counts is typically employed .

What are the considerations for studying CXCR7 in neurodevelopment and neuroprotection?

CXCR7 plays significant roles in neural progenitor migration and synaptic signaling, making it a valuable target in neuroscience research . When investigating CXCR7 in neural tissues:

• For immunohistochemistry in brain tissue, optimal results are typically achieved with TE buffer (pH 9.0) for antigen retrieval

• Consider the developmental stage of the tissue, as CXCR7 expression patterns change throughout neurodevelopment

• Examine co-localization with neural progenitor markers or mature neuronal markers depending on research question

• Investigate CXCR7 expression changes in response to neural injury or ischemic conditions

Research has implicated CXCR7 in neurodegenerative diseases, where targeting this receptor could potentially enhance recovery from stroke and other ischemic injuries .

How can I design experiments to investigate CXCR7-CXCL12 signaling dynamics?

Investigating CXCR7-CXCL12 signaling requires specialized experimental approaches to capture the complex receptor dynamics:

• Receptor internalization assays: Utilize fluorescently labeled antibodies to track CXCR7 trafficking before and after CXCL12 stimulation

• Beta-arrestin recruitment assays: Measure CXCR7's association with beta-arrestin following ligand binding

• Scavenger function analysis: Assess CXCL12 degradation rates in the presence or absence of functional CXCR7

• Heterodimer formation: Investigate CXCR7-CXCR4 heterodimer formation using proximity ligation assays or FRET-based approaches

• Signaling pathway analysis: Examine downstream pathways including ERK, AKT, and MAP kinase activation patterns

When designing such experiments, account for the unique properties of CXCR7 as an atypical chemokine receptor that primarily functions as a scavenger rather than a classical G-protein coupled receptor.

What role does CXCR7 play in cardiovascular development and how can antibodies help investigate this?

CXCR7 is essential for cardiovascular development and repair processes, including endothelial cell migration and heart valve formation . Research has demonstrated upregulation of CXCR7 expression during tissue repair, positioning it as a focal point for therapeutic angiogenesis studies.

When investigating CXCR7 in cardiovascular tissues:

• Use appropriate cardiovascular-specific positive controls (e.g., endothelial cells)

• Perform co-staining with endothelial markers (CD31, VE-cadherin) to confirm cell-specific expression

• Consider developmental timing, as CXCR7 expression is dynamically regulated during heart development

• Analyze expression in both healthy tissues and in response to ischemic injury or other cardiovascular stressors

How can CXCR7 antibodies contribute to understanding immune regulation mechanisms?

CXCR7 plays a significant role in fine-tuning immune responses by modulating leukocyte trafficking and inflammation processes . This has important implications for research in autoimmune diseases and infectious disease.

For immunological investigations:

• Examine CXCR7 expression across different immune cell populations using flow cytometry with validated antibodies

• Investigate changes in expression during immune cell activation, differentiation, and migration

• Analyze CXCR7 function in chemokine gradient formation and maintenance

• Explore the interplay between CXCR7 and other chemokine receptors in coordinating immune cell trafficking

• Consider the impact of CXCR7 on CXCL12 availability for CXCR4-expressing immune cells

What are the recommended approaches for quantifying CXCR7 expression in different experimental contexts?

Accurate quantification of CXCR7 expression requires tailored approaches depending on the experimental system:

For Western blot analysis:

• Normalize CXCR7 band intensity to loading controls (β-actin, GAPDH)

• Include a standard curve of recombinant protein when possible

• Use digital image analysis software with appropriate background correction

For immunohistochemistry and immunofluorescence:

• Establish consistent image acquisition parameters

• Employ digital pathology tools for quantitative analysis

• Consider both staining intensity and percentage of positive cells

• Use the H-score or Allred scoring systems for semi-quantitative assessment

For flow cytometry:

• Report expression as mean fluorescence intensity (MFI)

• Include fluorescence minus one (FMO) controls

• Use standardized beads for instrument calibration

How should researchers interpret discrepancies between CXCR7 mRNA and protein expression data?

Discrepancies between CXCR7 mRNA and protein expression are common and may reflect important biological phenomena. When encountering such discrepancies:

• Consider post-transcriptional regulation (miRNAs, RNA-binding proteins)

• Evaluate protein stability and turnover rates

• Assess receptor internalization and trafficking

• Examine technical factors such as antibody sensitivity compared to mRNA detection methods

• Verify findings using multiple complementary techniques (e.g., Western blot, IHC, flow cytometry)

Conducting parallel analyses of mRNA (RT-qPCR, RNA-seq) and protein (antibody-based methods) can provide insight into the regulatory mechanisms controlling CXCR7 expression in different physiological and pathological contexts.