CXE11 Antibody

What is CXCL11 and what are its primary biological functions?

CXCL11, also known as I-TAC (Interferon-inducible T-cell Alpha Chemoattractant), SCYB11, or beta-R1, is a non-ELR CXC chemokine that plays critical roles in immune cell trafficking and inflammatory responses. The canonical CXCL11 protein consists of 94 amino acids (with a 21-amino acid signal sequence cleaved to form the mature 73-amino acid protein) and has a molecular weight of approximately 10.4 kDa .

CXCL11 functions primarily as a chemoattractant for IL-2 activated T cells expressing CXCR3, but does not attract freshly isolated T cells, neutrophils, or monocytes . At the molecular level, CXCL11 participates in CXCR3 chemokine receptor binding and contributes to G-protein coupled receptor (GPCR) signaling pathways .

What experimental applications are suitable for CXCL11 antibodies?

CXCL11 antibodies can be employed across multiple experimental platforms depending on the specific research questions being addressed:

• Western Blotting (WB): CXCL11 antibodies can detect the protein in cell lysates, particularly from cells treated with IFN-gamma, LPS, and protein transport inhibitors like Brefeldin A. Typical dilutions range from 1:500 to 1:2000, though this should be optimized for each experimental system .

• Immunohistochemistry (IHC): These antibodies can detect CXCL11 in tissue sections, including cancer tissues such as human colon cancer. Recommended dilutions typically range from 1:50 to 1:500, with antigen retrieval using TE buffer (pH 9.0) or citrate buffer (pH 6.0) .

• Neutralization Assays: CXCL11 antibodies can block chemotaxis induced by recombinant CXCL11 in a dose-dependent manner, enabling functional studies of CXCL11-mediated cell migration. The neutralizing dose (ND50) is typically 0.5-1.5 μg/mL in the presence of 0.05 μg/mL recombinant human CXCL11 .

• Cell Signaling Studies: CXCL11 antibodies can be used to investigate downstream signaling events, such as CXCR7-mediated Rb degradation, providing insights into receptor-mediated signal transduction pathways .

How can I validate the specificity of a CXCL11 antibody?

Validating antibody specificity is crucial for ensuring reliable experimental results. For CXCL11 antibodies, consider the following validation strategies:

• Positive Control Selection: Use cells known to express CXCL11 after appropriate stimulation. THP-1 cells treated with IFN-gamma, LPS, and Brefeldin A represent a suitable positive control for Western blotting applications .

• Chemotaxis Neutralization Assay: Determine if the antibody can block CXCL11-induced chemotaxis in a dose-dependent manner. The BaF3 mouse pro-B cell line transfected with human CXCR3 provides a responsive system for measuring CXCL11-mediated chemotaxis. Effective neutralization indicates antibody specificity for biologically active CXCL11 .

• Cross-Reactivity Testing: Assess potential cross-reactivity with structurally related chemokines, particularly IP-10 and MIG, which share 36% and 37% amino acid sequence homology with CXCL11, respectively . This can be performed by comparing neutralization capacity against these related chemokines.

• Knockout/Knockdown Validation: Evaluate antibody signal in CXCL11 knockout or knockdown samples compared to wild-type controls. The absence or significant reduction of signal in these negative controls strongly supports antibody specificity .

What tissues and cell types express CXCL11, and how is this relevant for experimental design?

CXCL11 exhibits tissue-specific expression patterns that should inform experimental design:

• Basal Expression: Under normal conditions, CXCL11 shows low-level expression in thymus, spleen, pancreas, liver, lung, placenta, prostate, and intestine .

• Inducible Expression: CXCL11 expression is dramatically upregulated in astrocytes stimulated with IFN-gamma and IL-1. Moderate increases are also observed in stimulated monocytes .

• Pathological Expression: Elevated CXCL11 expression has been observed in various pathological conditions, including cancer tissues such as human colon cancer and gliomas .

When designing experiments to study CXCL11, researchers should consider:

• Using appropriate stimuli (particularly IFN-gamma) to induce expression in cell culture models

• Including relevant tissue controls based on known expression patterns

• Considering temporal dynamics of expression, as CXCL11 is often rapidly upregulated following stimulation

• Utilizing protein transport inhibitors (e.g., Brefeldin A) when detecting intracellular CXCL11 by Western blot

What is the optimal storage and handling protocol for CXCL11 antibodies?

Proper storage and handling are essential for maintaining antibody functionality and experimental reproducibility:

How can I design experiments to study CXCL11-mediated chemotaxis using neutralizing antibodies?

Designing robust chemotaxis experiments requires careful consideration of multiple parameters:

• Cell Model Selection: The BaF3 mouse pro-B cell line transfected with human CXCR3 provides a well-established model for studying CXCL11-mediated chemotaxis. This cell line demonstrates dose-dependent migration in response to recombinant human CXCL11 .

• Experimental Setup:

  • Use a two-chamber chemotaxis system (such as Transwell)

  • Place recombinant CXCL11 (typically at 0.05 μg/mL) in the lower chamber

  • Add cells to the upper chamber

  • For neutralization, pre-incubate CXCL11 with increasing concentrations of anti-CXCL11 antibody (0.5-1.5 μg/mL)

  • Include appropriate controls: no chemokine, chemokine only, and isotype control antibody

• Quantification Method: Cell migration can be quantified using viability dyes such as Resazurin, which provides a metabolic readout proportional to the number of migrated cells .

• Data Analysis:

  • Calculate the chemotactic index (ratio of cells migrating in response to chemokine versus random migration)

  • Determine the neutralizing dose (ND50), defined as the antibody concentration that reduces chemotaxis by 50%

  • Plot dose-response curves for both CXCL11-induced chemotaxis and antibody-mediated neutralization

• Validation Controls:

  • Test antibody specificity by examining its ability to neutralize chemotaxis induced by related chemokines (IP-10, MIG)

  • Verify that the antibody does not affect cell viability, which could confound migration results

What methodological approaches can be used to investigate CXCL11's role in immune cell recruitment during inflammatory responses?

Investigating CXCL11's role in immune recruitment requires integrating multiple experimental approaches:

• Transcriptional Analysis: Use RNA sequencing or qPCR to assess CXCL11 expression changes during inflammation. Single-cell RNA sequencing can further reveal cell type-specific expression patterns and regulatory networks .

• Protein-Protein Interaction Networks: Employ STRING network analysis to identify protein-protein interactions involving CXCL11. This approach can reveal functional clusters related to immune cell chemotaxis, chemokine-mediated signaling, and cellular responses to interferons .

• Gene Set Enrichment Analysis (GSEA): Apply GSEA to transcriptome data to identify pathways enriched in inflammatory conditions, such as cellular response to type II interferon (GO:0034341) and lymphocyte chemotaxis (GO:0048247) .

• In Vivo Neutralization Studies: Administer neutralizing CXCL11 antibodies in animal models of inflammation to assess effects on:

  • Immune cell recruitment (measured by flow cytometry)

  • Inflammatory marker expression (assessed by qPCR or protein arrays)

  • Histopathological changes (evaluated by immunohistochemistry)

• Differential Gene Expression Analysis: Compare transcriptional profiles before and after antibody treatment to identify:

  • Genes directly affected by CXCL11 neutralization

  • Secondary effects on downstream signaling pathways

  • Potential off-target effects of antibody treatment

This multi-faceted approach can provide comprehensive insights into CXCL11's role in inflammatory processes and potential therapeutic targets.

How do I analyze potential cross-reactivity between CXCL11 antibodies and other CXC chemokines?

Analyzing cross-reactivity requires systematic assessment of antibody specificity against structurally related proteins:

• Sequence Homology Analysis:

  • CXCL11 shares 36% and 37% amino acid sequence homology with IP-10 and MIG, respectively

  • Identify regions of highest homology, which represent potential cross-reactive epitopes

  • Compare the immunogen sequence used to generate the antibody with homologous regions in related chemokines

• ELISA-Based Cross-Reactivity Testing:

  • Coat plates with equal amounts of recombinant CXCL11, IP-10, MIG, and other CXC chemokines

  • Incubate with serial dilutions of the CXCL11 antibody

  • Develop and quantify binding signals

  • Calculate relative binding affinities to assess cross-reactivity

• Western Blot Analysis:

  • Run purified recombinant chemokines on SDS-PAGE

  • Transfer to membrane and probe with the CXCL11 antibody

  • Compare band intensities at equivalent protein loading

  • Include positive and negative controls to establish specificity thresholds

• Functional Neutralization Comparison:

  • Test the antibody's ability to neutralize biological activities of related chemokines

  • Compare neutralization potencies (ND50 values) across different chemokines

  • Significant differences in neutralization capacity indicate preferential specificity

• Epitope Mapping:

  • Use peptide arrays covering overlapping regions of CXCL11 and related chemokines

  • Probe arrays with the antibody to identify specific binding epitopes

  • Determine if the recognized epitopes are unique to CXCL11 or shared with other chemokines

What techniques can be used to investigate the interaction between CXCL11 antibodies and their target in different experimental contexts?

Understanding antibody-antigen interactions across experimental contexts requires specialized techniques:

• Surface Plasmon Resonance (SPR):

  • Immobilize CXCL11 or the antibody on a sensor chip

  • Measure real-time binding kinetics (kon and koff rates)

  • Determine equilibrium dissociation constant (KD)

  • Compare binding parameters under different buffer conditions to assess pH or salt sensitivity

• Immunoprecipitation (IP) Coupled with Mass Spectrometry:

  • Use CXCL11 antibodies to pull down the target protein from complex biological samples

  • Analyze precipitated proteins by mass spectrometry

  • Identify potential co-precipitating binding partners

  • Quantify enrichment of CXCL11 versus other proteins as a measure of specificity

• Immunohistochemistry Optimization:

  • Test multiple antigen retrieval methods (TE buffer pH 9.0 vs. citrate buffer pH 6.0)

  • Compare staining patterns across different tissue types

  • Include biological controls (tissues with known expression levels)

  • Perform blocking experiments with recombinant CXCL11 to confirm staining specificity

• Biolayer Interferometry (BLI):

  • Immobilize antibodies on biosensors

  • Measure binding to CXCL11 in real-time

  • Determine binding parameters in different buffer conditions

  • Assess competition with other ligands or receptors

• Epitope Binning:

  • Use competitive binding assays to classify antibodies into bins that recognize overlapping or distinct epitopes

  • Identify antibodies that compete with CXCR3 binding, which may have neutralizing potential

  • Map the relationship between epitope recognition and functional neutralization

How can CXCL11 antibodies be utilized to investigate interferon-responsive gene networks in disease models?

CXCL11 is strongly induced by interferons, making it a valuable marker for investigating interferon-responsive networks:

• Transcriptional Network Analysis:

  • Use CXCL11 antibodies to detect protein-level changes following interferon stimulation

  • Correlate these changes with transcriptional data from RNA sequencing

  • Apply network analysis to identify co-regulated genes in the interferon response pathway

  • Characterize temporal dynamics of the interferon response using time-course experiments

• Chromatin Immunoprecipitation (ChIP):

  • Use antibodies against transcription factors (e.g., STAT1) that regulate CXCL11 expression

  • Identify binding sites in the CXCL11 promoter and other interferon-stimulated genes

  • Construct regulatory networks based on shared transcription factor binding patterns

  • Correlate these networks with protein expression patterns detected by CXCL11 antibodies

• Single-Cell Analysis:

  • Combine CXCL11 immunostaining with other markers of interferon response

  • Use flow cytometry or imaging cytometry to classify cells based on their response patterns

  • Identify cell populations with differential interferon sensitivity

  • Correlate cellular heterogeneity with disease progression or treatment response

• Pathway Perturbation Experiments:

  • Apply neutralizing CXCL11 antibodies to disrupt specific nodes in the interferon network

  • Measure downstream effects on Gene Ontology pathways like "cellular response to type II interferon" (GO:0034341) and "lymphocyte chemotaxis" (GO:0048247)

  • Identify feedback mechanisms and compensatory responses

  • Construct predictive models of network behavior following therapeutic intervention

• Disease Model Applications:

  • Use CXCL11 antibodies as tools to modulate interferon-driven inflammation in disease models

  • Monitor effects on both transcriptional networks and clinical outcomes

  • Identify potential therapeutic targets within the interferon response network

  • Evaluate the translational potential of targeting CXCL11 in interferon-mediated diseases

How should I interpret discrepancies in CXCL11 detection between different antibody-based methods?

When facing inconsistent results across different detection methods, consider these analytical approaches:

• Method-Specific Limitations:

  • Western blotting: Denaturating conditions may destroy conformational epitopes

  • IHC: Fixation and antigen retrieval can affect epitope accessibility

  • ELISA: Native protein conformation is maintained, but may not reflect in vivo complexity

  • Flow cytometry: Cell permeabilization methods influence intracellular detection

• Systematic Validation Process:

  • Compare results across multiple antibody clones targeting different epitopes

  • Verify findings using orthogonal detection methods (e.g., mass spectrometry)

  • Test antibody performance in samples with known CXCL11 expression levels

  • Consider differences in detection sensitivity between methods

• Sample Preparation Variables:

  • For Western blots: Test different lysis buffers and protein extraction methods

  • For IHC: Compare multiple fixation protocols and antigen retrieval techniques (TE buffer pH 9.0 vs. citrate buffer pH 6.0)

  • For all methods: Assess the impact of sample storage and freeze-thaw cycles

• Data Integration Strategy:

  • Develop a weighted evidence approach that considers the strengths and limitations of each method

  • Prioritize functional validation (e.g., neutralization assays) over purely descriptive methods

  • Create a composite measure that integrates results across multiple detection platforms

What controls and experimental design considerations are essential when using CXCL11 antibodies in complex disease models?

Robust experimental design requires careful consideration of controls and potential confounding factors:

• Essential Controls:

  Control Type	Purpose	Implementation
  Positive Control	Verify antibody functionality	IFN-gamma/LPS-treated cells known to express CXCL11
  Negative Control	Assess background and non-specific binding	Isotype-matched irrelevant antibody
  Biological Negative	Confirm specificity	CXCL11 knockout/knockdown samples
  Absorption Control	Validate epitope specificity	Pre-incubate antibody with recombinant CXCL11
  Technical Replicate	Evaluate method reproducibility	Repeat assays with identical samples
  Biological Replicate	Account for biological variability	Independent samples from different subjects/animals

• Disease Model Considerations:

  • Temporal dynamics: Determine optimal sampling timepoints based on disease progression

  • Spatial heterogeneity: Account for regional variations in CXCL11 expression

  • Treatment effects: Consider how interventions might alter CXCL11 expression independently of disease processes

  • Comorbidities: Control for conditions that might influence CXCL11 expression

• Statistical Design Elements:

  • Power analysis: Determine appropriate sample sizes based on expected effect sizes

  • Blinding: Implement observer blinding to prevent unconscious bias

  • Randomization: Randomly assign subjects to experimental groups

  • Data normalization: Select appropriate housekeeping genes or proteins for normalization

How can I differentiate between direct and indirect effects when using CXCL11 neutralizing antibodies in biological systems?

Distinguishing direct from indirect effects requires systematic experimental approaches:

• Temporal Analysis:

  • Conduct time-course experiments to establish the sequence of molecular events

  • Direct effects typically occur rapidly after antibody administration

  • Indirect effects emerge later as downstream consequences

• Dose-Response Relationships:

  • Plot dose-response curves for various outcomes following antibody treatment

  • Direct effects often show similar dose-response relationships

  • Indirect effects may exhibit different dose thresholds or kinetics

• Pathway Inhibition Approach:

  • Combine CXCL11 neutralization with inhibitors of potential downstream pathways

  • If inhibiting a pathway blocks an effect of CXCL11 neutralization, that effect likely depends on the pathway

  • This approach can map the connectivity between direct and indirect effects

• Receptor Dependency:

  • Compare effects of CXCL11 neutralization with CXCR3 antagonism

  • Effects that occur with both interventions likely depend on CXCL11-CXCR3 signaling

  • Effects unique to CXCL11 neutralization may involve alternative receptors or non-canonical functions

• Transcriptional Network Analysis:

  • Identify early response genes following CXCL11 neutralization

  • Distinguish primary response genes (direct effects) from secondary response genes (indirect effects)

  • Construct network models that predict propagation of signals through cellular pathways

What factors influence the choice between polyclonal and monoclonal CXCL11 antibodies for specific research applications?

Selecting the appropriate antibody type depends on experimental goals and technical considerations:

• Application-Specific Considerations:

  Application	Polyclonal Advantages	Monoclonal Advantages
  Western Blotting	Higher sensitivity due to multiple epitope recognition	Higher specificity and batch consistency
  IHC	Better signal amplification in low-expression contexts	More consistent staining patterns across experiments
  Neutralization	May block multiple functional domains	Precise targeting of specific functional epitopes
  Flow Cytometry	Less affected by epitope masking	Cleaner background and more precise quantification

• Technical Factors:

  • Epitope accessibility: Polyclonals recognize multiple epitopes, increasing detection probability in partially denatured samples

  • Batch consistency: Monoclonals provide higher reproducibility across production lots

  • Cross-reactivity risk: Monoclonals typically offer higher specificity for closely related proteins

  • Signal-to-noise ratio: Application-dependent, with monoclonals generally providing cleaner backgrounds

• Experimental Variables:

  • Sample type: Formalin-fixed tissues may benefit from polyclonals due to epitope diversity

  • Antigen abundance: Low-abundance proteins may be better detected with polyclonals

  • Specificity requirements: Studies of highly homologous proteins benefit from monoclonal specificity

  • Long-term reproducibility needs: Extended studies require the consistency of monoclonals

• Research Stage Considerations:

  • Early exploratory research: Polyclonals provide broader detection capability

  • Validation studies: Parallel testing with both antibody types increases confidence

  • Standardized assays: Monoclonals ensure consistent performance across laboratories

How can transcriptomic data be integrated with CXCL11 antibody-based protein detection to enhance research findings?

Integrating transcriptomic and protein-level data provides a more comprehensive understanding of CXCL11 biology:

• Multi-omics Data Integration Framework:

  • Correlate CXCL11 mRNA expression from RNA-seq with protein levels detected by antibodies

  • Identify discordant patterns suggesting post-transcriptional regulation

  • Apply computational methods to model the relationship between transcription, translation, and protein stability

  • Use network analysis to place CXCL11 within larger regulatory frameworks

• Single-Cell Multi-omics Approach:

  • Combine single-cell RNA sequencing with protein detection (e.g., CITE-seq)

  • Map cellular heterogeneity in CXCL11 expression at both RNA and protein levels

  • Identify cell populations with differential regulation of CXCL11

  • Correlate cell state transitions with changes in CXCL11 expression

• Temporal Dynamics Analysis:

  • Track changes in CXCL11 mRNA and protein following stimulation

  • Determine time lags between transcriptional and translational responses

  • Identify regulatory mechanisms affecting the kinetics of CXCL11 expression

  • Model feedback circuits controlling CXCL11 production and function

• Functional Enrichment Strategy:

  • Perform Gene Ontology enrichment analysis on transcriptomic data

  • Compare enriched pathways (e.g., "lymphocyte chemotaxis" GO:0048247) with functional effects of CXCL11 neutralization

  • Identify convergent evidence from transcript-level and protein-level studies

  • Develop integrated hypotheses that explain observations across different data types

• Translational Research Applications:

  • Correlate CXCL11 expression patterns with clinical outcomes in disease models

  • Develop biomarker panels combining transcriptomic signatures with protein detection

  • Identify patient subgroups likely to respond to interventions targeting CXCL11

  • Guide therapeutic development based on integrated mechanistic understanding