BRAK Human, His

What is BRAK (CXCL14) and what are its alternative nomenclatures?

BRAK, officially designated as CXCL14, is a member of the CXC chemokine family. The name "BRAK" stands for Breast and Kidney-expressed chemokine, reflecting its preferential expression pattern. Other synonyms include NJAC and bolekine. BRAK is a divergent CXC chemokine that was initially identified through its preferential expression in normal versus malignant cells . As a chemokine, it belongs to a family of related proteins that regulate leukocyte infiltration into inflamed tissue and play significant roles in various disease processes .

What are the basic structural characteristics of human BRAK protein?

Human BRAK (CXCL14) is a 9.4 kDa protein that belongs to the CXC chemokine family. The recombinant form expressed in E. coli systems is typically produced with high purity (≥98% by SDS-PAGE and HPLC analysis) . The protein maintains specific structural features characteristic of CXC chemokines, including conserved cysteine residues that form disulfide bonds critical for its three-dimensional structure and biological activity.

How does the expression pattern of BRAK differ between normal and pathological tissues?

BRAK exhibits preferential expression in normal tissues compared to malignant cells, making it unique among chemokines . Research has demonstrated that BRAK is normally expressed in breast and kidney tissues, as implied by its name. This distinctive expression pattern suggests potential tumor suppressor functions, contrasting with many other chemokines that are often upregulated in malignant settings. This differential expression makes BRAK an interesting target for research on cancer progression and potential therapeutic applications.

What are the primary research applications for human recombinant BRAK protein?

Human recombinant BRAK protein is primarily used in functional studies investigating chemokine signaling pathways, immunomodulation, and cancer research. With a potency range of 1.0-10.0 ng/mL , it serves as a valuable tool for studying:

• Chemotactic responses in immune cells

• Cell migration and invasion assays

• Receptor binding studies with CXCR4 or other potential receptors

• Immunomodulatory effects in various cell types

• Tumor suppression mechanisms

• Inflammatory pathway investigations

How can BRAK be incorporated into cancer research protocols?

Given BRAK's observed differential expression between normal and malignant cells , researchers can integrate human recombinant BRAK protein into cancer research through several approaches:

• Expression profiling studies: Comparing BRAK expression across tumor types and stages versus normal tissues

• Functional assays: Testing the effects of exogenous BRAK on cancer cell proliferation, migration, and invasion

• Signaling analysis: Investigating downstream pathways affected by BRAK in cancer cells

• Tumor microenvironment studies: Examining how BRAK influences immune cell recruitment and function within the tumor microenvironment

• Therapeutic potential evaluation: Testing BRAK as a potential anti-cancer agent alone or in combination with established therapies

What cell culture models are most appropriate for studying BRAK functions?

The recombinant BRAK protein is certified as suitable for cell culture applications . Researchers should consider the following models:

• Immune cell models: Primary human monocytes, dendritic cells, or cell lines like THP-1 to study chemotactic functions

• Cancer cell models: Both BRAK-expressing and BRAK-deficient cancer cell lines to examine tumor suppressive effects

• Epithelial cell models: Breast and kidney cell lines, reflecting BRAK's natural expression sites

• Co-culture systems: Combining immune and cancer cells to study interactions in the presence of BRAK

• 3D culture models: For more physiologically relevant analysis of BRAK's influence on cell migration and tissue architecture

What is the optimal reconstitution procedure for lyophilized BRAK protein?

Proper reconstitution of lyophilized BRAK protein is critical for maintaining its biological activity. The recommended protocol includes:

• Brief centrifugation of the vial to collect all material at the bottom

• Reconstitution in sterile water to a concentration of 0.1-1.0 mg/mL

• Gentle mixing by slow rotation until completely dissolved

• Avoiding vigorous shaking or vortexing that might cause protein denaturation

• Filtration through a 0.22 μm filter for sterility if needed for cell culture

• Aliquoting to avoid repeated freeze-thaw cycles

• Storage of reconstituted protein at -20°C or -80°C for longer term

The reconstituted protein should be handled with sterile technique to prevent contamination, particularly for cell culture applications .

How should researchers optimize dosage and treatment times for BRAK in functional studies?

When designing experiments with BRAK protein, researchers should consider:

• Dose-response testing: Starting with the recommended potency range (1.0-10.0 ng/mL) but extending beyond this range (0.1-100 ng/mL) to establish full dose-response curves

• Time-course analysis: Evaluating both acute (minutes to hours) and chronic (days) responses

• Cell type considerations: Different cell types may require different concentrations for optimal response

• Medium composition: Testing activity in serum-free versus serum-containing media, as serum proteins may affect BRAK activity

• Receptor saturation analysis: Determining concentrations needed for half-maximal and maximal receptor occupation

What quality control measures should be implemented when working with recombinant BRAK?

To ensure experimental reproducibility and reliability, researchers should implement the following quality control measures:

• Purity verification: Confirm ≥98% purity via SDS-PAGE before experiments

• Endotoxin testing: Verify endotoxin levels are <0.1 EU/μg as specified , particularly important for immunological studies

• Activity validation: Perform chemotaxis assays with responsive cells to confirm biological activity

• Protein concentration verification: Use quantitative methods (BCA or Bradford assays) to verify protein concentration after reconstitution

• Batch consistency evaluation: When changing lots, perform comparative analysis to ensure consistent activity

How does BRAK interact with other chemokine systems in complex immunological environments?

Advanced research on BRAK should consider its place within the broader chemokine network:

• Receptor cross-talk: Investigating how BRAK signaling influences or is influenced by other chemokine receptors

• Synergistic or antagonistic effects: Examining combined effects of BRAK with other immunomodulatory factors

• Chemokine gradient modeling: Studying how BRAK contributes to complex chemotactic gradients in tissue microenvironments

• Receptor desensitization: Analyzing how BRAK exposure affects subsequent responses to other chemokines

• Computational modeling: Developing in silico models to predict BRAK's role in complex immunological networks

What are the challenges in studying BRAK's potential role in disease pathogenesis?

Researchers face several challenges when investigating BRAK's contributions to disease mechanisms:

• Expression variability: BRAK expression differs significantly across tissues and disease states

• Receptor complexity: Potential interactions with multiple receptors beyond currently identified ones

• Contextual activity: BRAK's effects may vary drastically depending on the cellular and molecular context

• Technical limitations: Detection of BRAK at physiologically relevant concentrations can be technically challenging

• Translational barriers: Bridging findings from recombinant protein studies to in vivo disease mechanisms requires careful experimental design

How can researchers effectively analyze BRAK's signaling pathways?

To comprehensively investigate BRAK signaling mechanisms:

• Phosphoproteomic analysis: Use mass spectrometry-based approaches to identify phosphorylation events triggered by BRAK

• Transcriptomic profiling: Employ RNA-seq to characterize gene expression changes following BRAK treatment

• Receptor identification: Utilize receptor blocking antibodies, competitive binding assays, and genetic knockdowns to confirm receptor specificity

• Pathway inhibitor screening: Systematically test inhibitors of major signaling pathways to delineate BRAK's signaling cascade

• Real-time signaling visualization: Implement FRET-based biosensors to monitor signaling events in live cells

What are common causes of reduced BRAK activity in experimental settings?

Researchers may encounter reduced BRAK activity due to:

• Protein degradation: Improper storage or excessive freeze-thaw cycles

• Adsorption issues: Protein binding to tubes or other surfaces during handling

• Receptor downregulation: Target cells becoming unresponsive due to receptor internalization

• Interfering factors: Presence of inhibitory substances in the experimental system

• Structural modifications: Chemical or physical modifications affecting protein conformation

To maintain optimal activity, store reconstituted BRAK at -20°C in suitable low-binding tubes and minimize freeze-thaw cycles .

How can researchers distinguish between specific and non-specific effects of BRAK in cell-based assays?

To ensure observed effects are specifically attributable to BRAK:

• Heat-inactivated controls: Compare with heat-denatured BRAK protein

• Antibody neutralization: Use anti-BRAK neutralizing antibodies to block specific effects

• Receptor antagonists: Include specific antagonists of known BRAK receptors

• Dose-dependency: Establish clear dose-response relationships

• Genetic approaches: Employ receptor knockdown/knockout models to confirm specificity

• Structurally related controls: Test other chemokines with similar structural properties but different specificities

What considerations are important when designing immunoassays to detect endogenous BRAK?

When developing or selecting immunoassays for BRAK detection:

• Antibody specificity: Validate antibodies against recombinant BRAK and confirm minimal cross-reactivity with other chemokines

• Detection limits: Ensure assay sensitivity is appropriate for expected physiological concentrations

• Matrix effects: Validate the assay in relevant biological matrices (serum, tissue lysates)

• Sample processing: Optimize sample collection and processing to preserve BRAK integrity

• Quantification standards: Use the same recombinant BRAK standard for calibration to ensure accurate quantification

How might comparative studies between BRAK and other chemokines advance our understanding of chemokine biology?

Comparative approaches can yield valuable insights:

• Structure-function correlations: Analyzing structural differences between BRAK and other chemokines to identify unique functional domains

• Evolutionary conservation: Examining conservation across species to identify critical functional regions

• Receptor promiscuity patterns: Comparing receptor binding profiles to understand chemokine-receptor recognition principles

• Pharmacological responses: Evaluating differential responses to inhibitors or enhancers across chemokine family members

• Disease associations: Systematically comparing disease associations to identify unique versus shared pathological mechanisms

What novel methodologies might enhance research on BRAK's biological functions?

Emerging technologies that could advance BRAK research include:

• CRISPR-based screening: Identifying genes that modulate BRAK response through genome-wide screens

• Single-cell analysis: Characterizing heterogeneous responses to BRAK at single-cell resolution

• Organ-on-chip platforms: Studying BRAK function in more physiologically relevant microenvironments

• Advanced imaging: Utilizing super-resolution microscopy to visualize BRAK-receptor interactions

• Computational modeling: Developing machine learning approaches to predict BRAK interactions and functions

How can systems biology approaches enhance our understanding of BRAK in health and disease?

Integrative approaches to BRAK biology should consider:

• Multi-omics integration: Combining genomic, transcriptomic, proteomic, and metabolomic data to build comprehensive models of BRAK function

• Network analysis: Mapping BRAK within broader signaling and regulatory networks

• Temporal dynamics: Capturing time-dependent changes in BRAK signaling through kinetic modeling

• Patient stratification: Identifying patient subgroups where BRAK signaling plays distinct roles

• Pharmacological targeting: Developing predictive models for therapeutic approaches targeting BRAK pathways