Recombinant Rat BRAK protein (Cxcl14)

What is Recombinant Rat BRAK (CXCL14) protein and how does it relate to other chemokines?

Recombinant Rat BRAK/CXCL14 is a non-glycosylated polypeptide chemokine belonging to the CXC chemokine superfamily. It is also known by several other names including MIP-2 gamma, KEC (kidney-expressed chemokine), and BMAC (B cell and monocyte-activating chemokine) . Unlike other CXC chemokines such as MIP-2, CXCL14 lacks the ELR domain that typically precedes the CXC motif, which contributes to its unique functional properties . When produced recombinantly in E. coli expression systems, Rat CXCL14 consists of a single polypeptide chain containing 77 amino acids with a molecular mass of approximately 9.4 kDa .

The mature rat CXCL14 protein shares high sequence homology with human and mouse variants, with mouse and human CXCL14 differing by only 2 residues, indicating strong evolutionary conservation of this protein . This conservation suggests crucial biological functions that have been maintained across species.

What are the optimal storage and handling conditions for Recombinant Rat CXCL14?

For maintaining optimal stability and activity of Recombinant Rat CXCL14, researchers should follow these evidence-based storage protocols:

• Lyophilized protein:

  • Store desiccated below -18°C

  • While stable at room temperature for approximately 3 weeks, long-term storage requires freezing temperatures

• Reconstituted protein:

  • Reconstitute in sterile 18MΩ-cm H₂O at a concentration not less than 100 μg/ml

  • Short-term storage (2-7 days): 4°C

  • Long-term storage: below -18°C

  • Avoid freeze-thaw cycles as they significantly reduce protein activity

Reconstitution should be performed using sterile technique, and the reconstituted protein can be further diluted into other aqueous solutions for experimental applications.

How can researchers verify the biological activity of Recombinant Rat CXCL14?

The primary biological activity of Rat CXCL14 can be verified through its chemoattractant properties for activated monocytes. Standard methodological approaches include:

• Chemotaxis assay: Using modified Boyden chambers or Transwell systems with a concentration range of 1.0-10.0 ng/ml of CXCL14 . Optimal results are observed when monocytes have been pre-treated with prostaglandin E₂ or forskolin, which are agents that activate specific signaling pathways .

• Cell migration tracking: Time-lapse microscopy to monitor directed cell movement in response to CXCL14 gradients.

• Calcium flux assay: Measuring intracellular calcium mobilization upon receptor binding, which indicates functional receptor-ligand interaction.

• Receptor binding assays: Utilizing labeled CXCL14 to measure direct binding to cell surface receptors.

Activity verification should include appropriate positive and negative controls, including known chemoattractants and chemokine receptor antagonists.

What expression systems are recommended for producing Recombinant Rat CXCL14?

E. coli expression systems represent the most widely used platform for producing Recombinant Rat CXCL14 . The T7 promoter system, particularly in pET vectors, is extremely effective for high-yield protein expression, potentially representing up to 50% of total cell protein in successful cases .

Key methodological considerations for optimal expression include:

• Promoter selection: The T7 promoter system is highly recommended for its efficiency and inducible expression capabilities .

• Bacterial strain selection: BL21(DE3) or derivatives carrying the λDE3 prophage encoding T7 RNA polymerase under transcriptional control of lacUV5 promoter .

• Expression control: Basal expression can be regulated through T7 lysozyme co-expression, which binds to T7 RNA polymerase and reduces background expression .

• Purification strategy: Proprietary chromatographic techniques are typically employed to achieve >95% purity as determined by RP-HPLC and SDS-PAGE analysis .

The non-glycosylated nature of CXCL14 makes bacterial expression suitable, as post-translational modifications are not required for basic functionality, though they may influence specific aspects of in vivo activity.

How does Rat CXCL14 compare functionally with mouse and human orthologs?

When planning experiments that extrapolate between species, researchers should consider:

• Receptor binding specificity: Small sequence variations may affect receptor binding affinity or specificity.

• Cell type responsiveness: Different species' cells may display varying sensitivity to CXCL14.

• Downstream signaling: Intracellular signaling cascade activation may differ slightly between species.

• Experimental validation: Cross-species activity should be empirically validated rather than assumed based on sequence similarity alone.

What methodological approaches are recommended for studying CXCL14's role in immunological processes?

CXCL14 exhibits selective chemoattractant properties for monocytes that have been treated with prostaglandin E₂ or forskolin , suggesting specialized roles in immunomodulation. To investigate these roles, the following methodological approaches are recommended:

• Monocyte activation studies:

  • Pre-treat monocytes with prostaglandin E₂ (1-10 μM) or forskolin (10-50 μM) for 2-4 hours

  • Assess migration response to CXCL14 concentration gradients (1.0-10.0 ng/ml)

  • Analyze changes in cell surface activation markers via flow cytometry

• Signaling pathway analysis:

  • Investigate the activation of specific signaling cascades (G-protein coupled receptor signaling, calcium mobilization, MAPK pathways)

  • Use selective inhibitors to identify critical pathway components

  • Employ phospho-specific antibodies to track signaling events via western blotting

• Gene expression profiling:

  • RNA-seq or microarray analysis of CXCL14-treated cells

  • qRT-PCR validation of differentially expressed genes

  • ChIP-seq to identify transcription factor binding events downstream of CXCL14 signaling

How can researchers distinguish between specific and non-specific effects when working with Recombinant Rat CXCL14?

To ensure experimental rigor and distinguish specific from non-specific effects, researchers should implement the following methodological controls:

• Dose-response relationships:

  • Test multiple concentrations of CXCL14 (0.1-100 ng/ml range recommended)

  • Establish clear dose-dependent effects

• Receptor antagonism:

  • Use specific receptor antagonists or blocking antibodies

  • Employ competitive inhibition with unlabeled chemokines

• Genetic approaches:

  • Utilize receptor knockout or knockdown models

  • Implement CRISPR-Cas9 editing to modify putative binding sites

• Specificity controls:

  • Include structurally similar but functionally distinct chemokines

  • Use heat-inactivated or enzymatically degraded CXCL14 as negative controls

• Source validation:

  • Verify protein purity (>95%) by RP-HPLC and SDS-PAGE

  • Confirm identity by mass spectrometry

What are common challenges in maintaining CXCL14 activity and how can they be addressed?

Several factors can compromise CXCL14 activity in experimental settings. Here are evidence-based strategies to address common challenges:

• Protein degradation:

  • Avoid repeated freeze-thaw cycles

  • Add protease inhibitors to working solutions

  • Prepare fresh dilutions for each experiment

  • Store aliquots rather than stock solutions

• Activity loss during reconstitution:

  • Use the recommended reconstitution buffer (sterile 18MΩ-cm H₂O)

  • Maintain minimum concentration (100 μg/ml) during reconstitution

  • Avoid vigorous agitation that may cause denaturation

  • Consider adding carrier proteins for very dilute solutions

• Inconsistent experimental results:

  • Standardize cell culture conditions

  • Control for cell passage number and density

  • Establish reproducible activation protocols for monocytes

  • Maintain consistent assay conditions (temperature, pH, ion concentrations)

How can the purity and identity of Recombinant Rat CXCL14 be verified in laboratory settings?

To ensure experimental reproducibility, verification of protein purity and identity is essential. Recommended methodological approaches include:

• Purity assessment:

  • RP-HPLC analysis (should show >95% purity)

  • SDS-PAGE with Coomassie or silver staining

  • Capillary electrophoresis

• Identity confirmation:

  • Western blot with specific anti-CXCL14 antibodies

  • Mass spectrometry analysis (MALDI-TOF or ESI-MS)

  • N-terminal sequencing

  • Peptide mapping

• Functional verification:

  • Chemotaxis assay using activated monocytes (1.0-10.0 ng/ml concentration range)

  • Receptor binding assays

What potential roles does CXCL14 play beyond its established function as a monocyte chemoattractant?

While the search results primarily highlight CXCL14's role in monocyte chemotaxis, emerging research suggests broader biological functions. Researchers investigating these areas should consider:

• Tissue distribution patterns:

  • CXCL14 is constitutively expressed at high levels in the basal layer of epidermal keratinocytes and dermal fibroblasts in skin tissues

  • Also expressed in lamina propria cells in normal intestinal tissues

  • This tissue-specific expression pattern suggests specialized roles in epithelial immunity and homeostasis

• Methodological approaches for exploring novel functions:

  • Tissue-specific knockout models

  • Single-cell RNA sequencing to identify CXCL14-responsive cell populations

  • Spatial transcriptomics to map expression patterns in complex tissues

  • Ex vivo tissue explant cultures to study physiological responses

How does CXCL14 interact with other components of the chemokine network?

Understanding CXCL14's place within the broader chemokine network requires specialized experimental approaches:

• Receptor sharing and competition studies:

  • Competition binding assays with other CXC chemokines

  • Receptor internalization and recycling studies

  • Heterologous desensitization experiments

• Synergy and antagonism assessment:

  • Combinatorial treatment with multiple chemokines

  • Analysis of receptor complex formation

  • Investigation of shared downstream signaling pathways

• In vivo interaction mapping:

  • Multiplex cytokine profiling in various physiological and pathological conditions

  • Chemokine gradient visualization in tissue sections

  • Real-time intravital imaging of leukocyte recruitment