Recombinant Mouse Stromal cell-derived factor 1 protein (Cxcl12), partial (Active)

What is the biological function of mouse CXCL12/SDF-1α?

Mouse CXCL12 (also known as SDF-1α) is a chemokine that primarily signals through the G protein-coupled receptor CXCR4 to recruit activated leukocytes . This protein plays crucial roles in embryogenesis, hematopoiesis, and angiogenesis, while also being implicated in pathological conditions including HIV-1, WHIM disease, and cancer progression . The biological importance of CXCL12 is demonstrated by its involvement in stem cell mobilization, tissue regeneration, and immune cell trafficking. In cardiovascular research, exogenous administration of recombinant SDF-1α has been shown to enhance neovasculogenesis and improve cardiac function following myocardial infarction .

How does mouse CXCL12 compare structurally to human CXCL12?

Mouse and human CXCL12 share remarkable sequence conservation with approximately 99% sequence identity . This high degree of conservation reflects the crucial evolutionary importance of this chemokine. Key structural elements include:

• N-terminal region: Critical for receptor activation and binding

• C-terminal region: Important for extracellular stabilization

• Dimerization interface: Involves specific residues (His25, Lys27, and Ala40) that are highly conserved between species

This structural similarity makes mouse CXCL12 an excellent model for studying human CXCL12-related processes, although minor species-specific differences should be considered when translating findings to human applications.

What methods are most effective for verifying recombinant mouse CXCL12 activity?

Multiple complementary approaches can be used to verify the activity of recombinant mouse CXCL12:

When verifying activity, researchers should select methods most relevant to their specific application while considering that different cellular contexts may yield varying responses to CXCL12 stimulation.

What are the optimal storage and handling conditions for recombinant mouse CXCL12?

For maximum stability and activity retention of recombinant mouse CXCL12:

• Storage temperature: Store lyophilized protein at 4-8°C for short-term (1 week) or at -20°C to -80°C for long-term storage

• Reconstitution method: Gently reconstitute by pipetting the recommended solution down the sides of the vial without vortexing

• Working solution preparation: For prolonged storage, dilute to working aliquots in a 0.1% BSA solution to stabilize the protein

• Freeze-thaw cycles: Aliquot reconstituted protein to avoid repeated freeze-thaw cycles that can significantly decrease activity

• Quality verification: Before experimental use, verify protein purity (>95% by SDS-PAGE) and confirm low endotoxin levels (<0.2 EU per 1 μg of protein)

Following these guidelines ensures maintained biological activity and experimental reproducibility when working with this sensitive chemokine.

How does the monomer-dimer equilibrium of CXCL12 affect its signaling properties?

The oligomeric state of CXCL12 significantly impacts its biological functions:

CXCL12 exists in a dynamic equilibrium between monomeric and dimeric forms, with the dimeric form becoming more prevalent at higher concentrations. This equilibrium has profound effects on signaling:

• Structural basis: Dimerization occurs via the β-strand core at the monomer-monomer interface, involving residues His25, Lys27, and Ala40

• Receptor interactions: Monomeric and dimeric CXCL12 engage CXCR4 differently, potentially activating distinct downstream signaling pathways

• Functional consequences: CXCL12 locked-dimer forms (CXCL12-LD) demonstrate partial agonist properties compared to wild-type CXCL12 but enhance CXCR4 internalization

• Research applications: This knowledge can be exploited to design engineered CXCL12 variants with specific oligomeric states for targeted applications

Researchers can manipulate this equilibrium experimentally by:

• Varying protein concentration: Higher concentrations favor dimer formation

• Buffer conditions: Changes in ionic strength, pH, or addition of small molecules like fosfosal can shift the equilibrium

• Engineered variants: Creating locked monomers or dimers through strategic mutations or chemical crosslinking

Understanding this equilibrium is crucial when designing experiments, as the predominant oligomeric state will influence experimental outcomes.

What are the optimal experimental models for studying CXCL12 function in cardiovascular research?

Cardiovascular research involving CXCL12 requires careful selection of experimental models:

In Vitro Models:

• Endothelial progenitor cell (EPC) migration assays using Boyden chambers to assess chemotactic potential

• Receptor activation studies quantifying phosphorylated AKT in cardiac cells following CXCL12 treatment

• Angiogenesis assays including tube formation by endothelial cells and angiopoietin-1 expression analysis

In Vivo Models:

• Myocardial infarction induced by left anterior descending coronary artery ligation in mice, followed by peri-infarct intramyocardial injection of CXCL12

• Functional assessment via echocardiography measuring ejection fraction, cardiac output, stroke volume, and fractional area change

• Molecular analysis of angiogenic factor expression in CXCL12-treated cardiac tissue

Advanced Approaches:

• Computational protein design to create engineered SDF analogs (ESA) with enhanced stability and function

• Integration of CXCL12 or its analogs with biomaterial delivery systems for sustained release in the cardiac microenvironment

• Multi-parametric assessment combining functional, molecular, and cellular readouts for comprehensive analysis

When selecting a model, researchers should consider the specific research question, available resources, and translational relevance of the chosen system.

How should researchers design CXCL12 chemotaxis assays for optimal results?

Designing robust CXCL12 chemotaxis assays requires attention to several critical parameters:

Technical Setup Considerations:

• Assay format selection: While Boyden chambers are standard, microfluidic devices may provide better gradient control for mechanistic studies

• Gradient establishment: CXCL12 concentration ranges typically span 10-1000 ng/mL, with optimal concentrations varying by cell type

• Membrane selection: Pore size should be appropriate for the cell type (typically 3-8 μm)

Experimental Controls:

• Positive control: Include a known chemotactic agent for your specific cell type

• Negative control: Use buffer-only conditions to establish baseline migration

• Specificity control: Include CXCR4 antagonists (like AMD3100) to confirm receptor dependence

Optimization Parameters:

Analysis Methods:

• Quantification: Count cells in multiple random fields per membrane or use fluorescence-based detection

• Data representation: Calculate chemotactic index (ratio of directed to random migration) rather than absolute cell numbers

• Statistical analysis: Use appropriate tests for multiple concentration comparisons (typically ANOVA with post-hoc tests)

Thorough optimization and standardization of these parameters are essential for generating reproducible and physiologically relevant chemotaxis data.

What considerations should be made when using recombinant mouse CXCL12 in in vivo versus in vitro studies?

The experimental context significantly affects how recombinant mouse CXCL12 should be employed:

Species Compatibility:

• Despite 99% sequence identity between mouse and human CXCL12 , species-specific variants should be matched to experimental systems

• Use mouse CXCL12 in mouse models and cell lines; human CXCL12 in human cells or humanized mouse models

Dosing Considerations:

Delivery Methods:

• In vitro: Direct addition to culture media with consideration of protein stability over time

• In vivo: Multiple options including intravenous, subcutaneous, or direct tissue injection (e.g., peri-infarct myocardial injection in cardiac studies )

Assessment of Outcomes:

• In vitro: Direct cellular responses (migration, signaling, gene expression) with clearly defined endpoints

• In vivo: More complex physiological outcomes requiring appropriate animal models and analytical techniques

Translational Considerations:

• Effects observed in simplified in vitro systems may not directly translate to complex in vivo environments

• Consider pharmacokinetics, biodistribution, and potential immunogenicity when designing in vivo studies

Researchers should always include appropriate controls and validation approaches specific to their experimental context.

How can recombinant mouse CXCL12 be used in stem cell mobilization studies?

Recombinant mouse CXCL12 plays a central role in stem cell biology and can be strategically employed in mobilization studies:

Experimental Design Approaches:

• Administration protocols: CXCL12 variants (particularly engineered forms like CXCL12-LD) can be administered systemically to evaluate stem cell mobilization into peripheral blood

• Comparative studies: Compare wild-type CXCL12, locked-monomer, and locked-dimer forms to elucidate optimal mobilization strategies

• Combinatorial approaches: Study CXCL12 in combination with other mobilizing agents (G-CSF, plerixafor) for potential synergistic effects

Assessment Methodologies:

• Flow cytometry analysis of stem cell markers (Sca-1, c-Kit, CD34) in peripheral blood at defined timepoints

• Colony-forming unit assays to evaluate functional properties of mobilized cells

• In vivo tracking using labeled cells to study homing and engraftment post-mobilization

Research Applications:

• Hematopoietic recovery models following myeloablative therapy

• Cardiovascular repair studies in myocardial infarction models

• Acute myelogenous leukemia research examining how CXCL12 dimer signaling modulates disease processes

Advanced Considerations:

• CXCL12 variants that enhance CXCR4 internalization may avoid pharmacodynamic tolerance, offering advantages in repeated mobilization protocols

• The balance between mobilization and retention signals is critical; CXCL12 gradients between bone marrow and blood require careful manipulation

This research area demonstrates how recombinant CXCL12's biological properties can be harnessed for therapeutic stem cell mobilization strategies.

How does phosphorylation of AKT relate to CXCL12-mediated cell signaling?

AKT phosphorylation represents a critical node in CXCL12 signal transduction:

Signaling Cascade Mechanics:

• CXCL12 binding to CXCR4 activates heterotrimeric G proteins (primarily Gαi)

• This activation triggers multiple downstream pathways, prominently PI3K

• PI3K generates phosphatidylinositol (3,4,5)-trisphosphate, recruiting AKT to the membrane

• AKT undergoes phosphorylation at two key residues:

  • Thr308 (by PDK1)

  • Ser473 (by mTORC2)

• Fully phosphorylated AKT mediates numerous cellular responses

Functional Outcomes:

• Cell survival through inhibition of pro-apoptotic factors and activation of anti-apoptotic mechanisms

• Cytoskeletal reorganization necessary for directional migration in response to CXCL12 gradients

• Cell cycle progression supporting proliferative responses

Experimental Assessment Approaches:

• Western blotting to quantify phosphorylated AKT (pAKT) relative to total AKT

• Time-course analysis (AKT phosphorylation typically occurs within minutes of CXCL12 stimulation)

• Inhibitor studies using PI3K inhibitors (LY294002, wortmannin) or AKT inhibitors to confirm pathway specificity

Comparative Analysis:
Engineered CXCL12 analogs like ESA have demonstrated enhanced efficiency in inducing AKT phosphorylation compared to native SDF-1α, correlating with improved functional outcomes in cardiovascular models .

Understanding this signaling node is crucial for developing targeted interventions that modulate specific aspects of CXCL12-mediated cellular responses.

How can engineered CXCL12 analogs be designed and what advantages do they offer?

Strategic protein engineering can produce CXCL12 variants with enhanced properties:

Design Strategies:

• Computational protein design using structure modeling to engineer SDF polypeptide analogs (like ESA)

• Domain splicing that combines the N-terminus (activation/binding) and C-terminus (stabilization) with structural elements maintaining their orientation

• Conformational constraints introduction (such as diproline segments) to limit peptide backbone flexibility while preserving native structure

• Oligomerization manipulation to create locked monomers or dimers with specific functional properties

Advantages of Engineered Analogs:

Validation Approaches:

• Comparative functional assays between engineered analogs and native CXCL12

• Structural confirmation using NMR spectroscopy or X-ray crystallography

• In vivo testing in relevant disease models to demonstrate improved outcomes

The engineered SDF analog (ESA) case study demonstrates the translational potential of this approach, showing superior performance in inducing EPC migration and improving post-myocardial infarction cardiac function compared to native SDF-1α .

What methodological approaches are most effective for studying CXCL12 in cancer research?

Cancer research involving CXCL12 employs diverse methodological approaches:

Expression Analysis Methods:

• Immunohistochemistry for spatial assessment of CXCL12 in tumor tissues

• ELISA/Quantikine assays for quantification in fluid samples (serum, plasma, CSF)

• Transcriptomic analysis through RT-PCR, microarray, or RNA-seq approaches

Prognostic Studies Design:

Functional Investigations:

• Cell migration/invasion assays examining CXCL12's effect on cancer cell metastatic potential

• Receptor activation studies assessing CXCR4 signaling in response to CXCL12 stimulation

• CXCR4 antagonist/inhibitor studies to evaluate therapeutic potential

Translational Research:

• Biomarker development validating CXCL12 as a diagnostic or prognostic indicator

• Receiver operating characteristic (ROC) analysis to determine diagnostic accuracy

• Development of combination biomarker panels incorporating CXCL12 with other markers

What are the technical challenges in measuring CXCL12 expression levels in biological samples?

Accurate CXCL12 quantification in biological samples faces several technical hurdles:

Sample Collection and Processing Challenges:

• Rapid degradation of CXCL12 in biological fluids necessitates immediate processing or preservation

• Platelet activation during blood collection can release CXCL12, potentially skewing measurements

• Standardized protocols are essential to minimize pre-analytical variability

Detection Method Considerations:

• Different methodologies (ELISA, mass spectrometry, Western blot) have varying sensitivity and specificity profiles

• Antibody cross-reactivity with other chemokines or CXCL12 isoforms can compromise specificity

• Calibration with appropriate standards is critical for reliable quantification

CXCL12 Isoform and State Challenges:

• Multiple splice variants (α, β, γ) may not be equally detected by all assays

• Post-translational modifications affect detection efficiency

• Oligomeric state (monomer vs. dimer) influences detection depending on assay principles

Biological Matrix Complications:

Method Validation Requirements:

• Thorough validation including linearity, recovery, precision, and specificity studies

• Establishment of appropriate reference ranges for different sample types

• Inter-laboratory standardization to facilitate result comparison across studies

In comparative studies between CSF and plasma CXCL12 measurements in ALS patients, CSF determination showed significantly higher accuracy (AUC=0.97) compared to plasma determination (AUC=0.65), highlighting the importance of sample type selection .

How does CXCL12 signaling differ between normal and pathological conditions?

CXCL12 signaling undergoes significant alterations in disease states:

Receptor Expression Dynamics:

• Normal conditions: CXCR4 expression is tightly regulated and restricted to specific cell types

• Pathological conditions: Many diseases exhibit CXCR4 overexpression, particularly in cancers, altering response magnitude to CXCL12

Signaling Characteristics:

• Normal conditions: Transient signaling controlled by receptor internalization and degradation

• Pathological conditions: Persistent signaling due to impaired regulatory mechanisms or continuous ligand production

Pathway Activation Patterns:

• Normal conditions: Balanced activation of downstream pathways (PI3K/AKT, MAPK, JAK/STAT)

• Pathological conditions: Skewed activation favoring specific pathways (e.g., enhanced survival signaling in cancer cells)

Disease-Specific Alterations:

Therapeutic Implications:

• Normal physiological functions must be preserved while targeting pathological CXCL12 signaling

• Engineered CXCL12 analogs or receptor-specific interventions offer promising approaches for modulating disease-specific alterations

Understanding these differences is crucial for developing targeted therapies that normalize disrupted CXCL12 signaling without compromising essential physiological functions.

What is the potential of CXCL12 as a biomarker in neurological disorders?

Recent research has revealed significant potential for CXCL12 as a biomarker in neurological conditions:

Amyotrophic Lateral Sclerosis (ALS):

• CSF CXCL12 levels are significantly elevated in ALS patients (797.07±31.84 pg/mL) compared to healthy controls (316.15±16.6 pg/mL) and ALS-mimic conditions

• Diagnostic accuracy demonstrates remarkable performance with ROC analysis yielding an AUC value of 0.97±0.012 (95% CI 0.95-0.9959)

• Optimal diagnostic cut-off determined at 568.3 pg/mL with 85.11% sensitivity and 98.53% specificity

• Particularly valuable for differential diagnosis between ALS and ALS-mimic conditions including myelopathy, inflammatory polyneuropathy, and other motor disorders

Comparative Biomarker Analysis:

• CSF CXCL12 shows comparable diagnostic accuracy to neurofilament light chain (NF-L) in distinguishing ALS from healthy controls

• CSF determination is significantly more accurate than plasma determination for CXCL12, while NF-L shows similar accuracy in both biofluids

• Combined biomarker panels incorporating CXCL12 with other markers may enhance diagnostic precision

Methodological Considerations:

• Sample collection standardization is crucial for accurate CXCL12 measurement

• Validated ELISA kits (such as Human CXCL12/SDF-1α Quantikine ELISA) provide reliable quantification

• Test performers should be blinded to clinical information to prevent bias

Future Research Directions:

• Longitudinal studies correlating CXCL12 levels with disease progression

• Exploration of CXCL12's potential in other neurological disorders

• Investigation of CXCL12 signaling as a therapeutic target in neuroinflammatory conditions

The exceptional performance of CXCL12 as a biomarker in ALS highlights its potential value in the challenging area of neurological disease diagnosis.

How can CXCL12-CXCR4 axis modulation be applied in cancer therapeutic strategies?

The CXCL12-CXCR4 axis offers multiple intervention points for cancer therapy:

Therapeutic Rationale:

Intervention Strategies:

Optimal Application Contexts:

• Metastasis prevention: Disrupting CXCL12 gradients that guide cancer cell migration

• Chemosensitization: Combining CXCR4 antagonists with conventional chemotherapy

• Cancer stem cell targeting: Addressing the CXCL12-dependent niche that maintains cancer stem cells

Emerging Approaches:

• CXCL12-LD partial agonists that enhance CXCR4 internalization may avoid pharmacodynamic tolerance issues seen with conventional antagonists

• Small molecules that disrupt CXCL12 dimerization (like fosfosal) represent a novel intervention strategy targeting the ligand rather than the receptor

Translational Considerations:

• Patient stratification based on tumor CXCL12 expression levels may identify those most likely to benefit from targeted therapies

• Monitoring CXCL12 levels during treatment could provide pharmacodynamic biomarkers of response

These strategies represent a promising frontier in precision oncology, targeting a pathway critically involved in cancer progression and treatment resistance.

What emerging technologies are advancing CXCL12 research?

Cutting-edge technologies are transforming how researchers study CXCL12:

Advanced Protein Engineering:

• Computational protein design creating optimized CXCL12 analogs with enhanced stability and function

• Structure-guided approaches for developing variants with selective signaling properties

• Protein splicing techniques combining functional domains with structural stabilizing elements

Novel Analytical Methods:

• Advanced NMR approaches for studying CXCL12-receptor interactions at atomic resolution

• Single-molecule fluorescence techniques to observe CXCL12-CXCR4 binding dynamics in real-time

• Single molecule array (SIMOA) technology enabling ultrasensitive detection of biomarkers in biofluids

Innovative Delivery Systems:

• Biomaterial platforms for controlled release of CXCL12 or its analogs

• Targeted nanoparticle approaches for tissue-specific delivery

• Cell-based systems secreting engineered CXCL12 variants for sustained local effects

Systems Biology Approaches:

• Multi-omics integration to comprehensively map CXCL12 signaling networks

• Computational modeling of CXCL12 gradient formation and cellular responses

• Machine learning applications for predicting patient responses to CXCL12-targeted interventions

Translational Technologies:

• Non-invasive imaging techniques for tracking CXCL12-mediated processes in vivo

• Biomarker panels combining CXCL12 with complementary markers for enhanced diagnostic accuracy

• Point-of-care diagnostic platforms for rapid CXCL12 quantification in clinical settings

These technological advances are accelerating both fundamental understanding of CXCL12 biology and the development of CXCL12-based therapeutic strategies.

What key questions remain unanswered in CXCL12 research?

Despite significant progress, several critical questions in CXCL12 research remain to be addressed:

Molecular Mechanisms:

• How do different oligomeric states of CXCL12 precisely activate distinct signaling pathways?

• What determines the specificity of CXCL12-mediated responses in different cell types?

• How do post-translational modifications of CXCL12 regulate its function in different physiological contexts?

Pathophysiological Roles:

• What mechanisms drive elevated CXCL12 expression in neurological disorders like ALS?

• How does the CXCL12-CXCR4 axis contribute to treatment resistance in cancer beyond cell migration?

• What is the precise role of CXCL12 in tissue-specific regeneration processes?

Therapeutic Development:

• Can CXCL12-targeted interventions avoid disrupting essential physiological functions?

• What are the optimal properties for engineered CXCL12 analogs for specific therapeutic applications?

• How can combination approaches targeting multiple aspects of CXCL12 signaling be optimized?

Clinical Translation:

• Which patient populations would benefit most from CXCL12-targeted therapies?

• What are the most reliable biomarkers for monitoring response to CXCL12-targeted interventions?

• How can the promising results from preclinical models be effectively translated to human patients?

Technical Challenges:

• How can we develop more specific methods for detecting different CXCL12 isoforms and oligomeric states?

• What approaches can overcome the challenges in measuring CXCL12 in different biological matrices?

• How can we better model the complex spatial and temporal dynamics of CXCL12 gradients in vivo?

Addressing these questions will require interdisciplinary approaches combining advanced technologies with rigorous experimental design.

How might CXCL12 research impact personalized medicine approaches?

CXCL12 research has significant implications for advancing personalized medicine:

Biomarker-Based Patient Stratification:

• Tumor CXCL12 expression levels can identify cancer patients with poorer prognosis who may benefit from more aggressive intervention

• CSF CXCL12 measurements enable differentiation between ALS and ALS-mimic conditions with high accuracy (AUC=0.97), allowing for more precise diagnosis and treatment planning

• CXCL12 signaling patterns may predict response to specific therapies, guiding treatment selection

Tailored Therapeutic Approaches:

• Engineered CXCL12 analogs can be designed for specific disease applications, such as cardiovascular repair after myocardial infarction

• CXCL12-LD partial agonists that enhance receptor internalization may be particularly valuable for certain conditions while avoiding pharmacodynamic tolerance

• Dosing and administration protocols can be optimized based on individual patient characteristics and disease manifestations

Precision Monitoring:

• Serial measurement of CXCL12 levels could provide personalized biomarkers for disease progression or treatment response

• Comparative analysis of CXCL12 levels in different biofluids (CSF vs. plasma) may offer insights into disease-specific pathophysiology

• Integration of CXCL12 data with other biomarkers can create personalized multi-parameter disease monitoring systems

Genetic and Molecular Profiling Integration:

• Combined analysis of CXCL12/CXCR4 genetic variants and expression patterns with broader molecular profiles

• Correlation of CXCL12 signaling with specific disease subtypes or endotypes

• Development of companion diagnostics for CXCL12-targeted therapies

Translational Roadmap:

• Development of standardized, clinically validated CXCL12 measurement methods

• Large-scale biomarker studies correlating CXCL12 with clinical outcomes

• Clinical trials of CXCL12-targeted therapies with patient stratification based on biomarker profiles

• Integration of CXCL12 assessment into clinical decision support systems

These approaches highlight how CXCL12 research is contributing to the broader shift toward more personalized and precise medical interventions.