Recombinant Mouse Protein C10 (Grcc10)

What is Recombinant Mouse Protein C10 and what are its alternative designations?

Recombinant Mouse Protein C10 (CCL6) is a small cytokine (approximately 11 kDa) belonging to the CC chemokine family that has been identified specifically in rodents. It is also known by several synonyms including Small inducible cytokine A6, CCL6, C10 protein, MRP-1, Scya6, and chemokine (C-C motif) ligand 6 . The recombinant form is typically produced in E. coli expression systems and comprises the functional region of the native protein.

What is the amino acid sequence of Recombinant Mouse C10 protein?

The amino acid sequence of Recombinant Mouse C10 protein is: GLIQEIEKED RRYNPPIIHQ GFQDTSSDCC FSYATQIPCK RFIYYFPTSG GCIKPGIIFI SRRGTQVCAD PSDRRVQRCL STLKQGPRSG NKVIA . Commercial preparations, such as those described in the search results, typically include the truncated form spanning amino acids Gly42-Ala116 of the native protein .

What is the primary biological activity of Recombinant Mouse C10?

The primary biological activity of Recombinant Mouse C10 is its chemotactic effect on specific cell types. It has been demonstrated to chemoattract human CCR1-transfected BaF3 mouse proB cells at a concentration range of 0.05-0.25 μg/ml . The ED50 (effective dose for 50% response) for this chemotactic activity has been measured at 0.5-2 ng/mL , indicating high potency for this specific biological function.

How should I design an experiment to evaluate the chemotactic activity of Recombinant Mouse C10?

When designing an experiment to evaluate the chemotactic activity of Recombinant Mouse C10, implement a controlled experimental design with systematic manipulation of the independent variable (C10 concentration). First, establish a clear hypothesis about the protein's effect on cell migration . Use a concentration gradient (typically 0.05-0.25 μg/ml) based on established ED50 values (0.5-2 ng/mL) . Include both negative controls (buffer without chemokine) and positive controls (known chemotactic factors).

For your experimental treatment, prepare a randomized block design grouping by cell type or receptor expression levels . Measure chemotaxis using established methods such as transwell migration assays, and quantify results through cell counting or fluorescent labeling. Consider both the number of migrating cells and their migration distance as dependent variables. Statistical analysis should include ANOVA to assess significance across different concentration groups.

What control conditions are essential when evaluating immune responses to Recombinant Mouse C10?

When evaluating immune responses to Recombinant Mouse C10, several critical control conditions must be incorporated:

• Negative control: Include a phosphate-buffered saline (PBS) group with the same adjuvant used for the experimental groups .

• Adjuvant control: Test the adjuvant alone to distinguish between adjuvant-induced and protein-specific responses.

• Related protein control: Include a structurally similar but functionally distinct chemokine to demonstrate specificity.

• Dose-dependent controls: Test multiple concentrations (typically 50-100 μg per mouse) to establish dose-response relationships .

• Timing controls: Evaluate responses at multiple time points post-immunization.

This approach, similar to the experimental design used for LpxC and GmhA proteins in immunization studies , allows for proper attribution of observed immunological effects specifically to the C10 protein.

What sample size considerations apply for mouse models testing Recombinant Mouse C10?

For mouse models testing Recombinant Mouse C10, sample size determination should balance statistical power requirements with ethical considerations of animal use. Based on comparable immunization studies, a minimum of 10 mice per experimental group is recommended to achieve adequate statistical power . This sample size has been shown to successfully detect protective effects of recombinant proteins in challenge studies where protection rates of 50-60% were observed .

The experimental design should incorporate randomization of subjects to treatment groups to minimize bias. Consider using a randomized block design, stratifying mice by factors such as age or weight before random assignment to treatment groups . Power analysis should be conducted prior to experimentation based on expected effect sizes from preliminary data or literature values to ensure adequate statistical power (typically 0.8 or greater).

What are the optimal reconstitution protocols for lyophilized Recombinant Mouse C10?

The optimal reconstitution protocols for lyophilized Recombinant Mouse C10 vary depending on the preparation format (with or without carrier protein). For carrier-containing preparations, reconstitute at 100 μg/mL in sterile PBS containing at least 0.1% human or bovine serum albumin . For carrier-free preparations, reconstitute at 100 μg/mL in sterile PBS without additional proteins .

Alternative reconstitution methods suggest using sterile 18MΩ-cm H₂O at a concentration not less than 100 μg/ml, which can then be further diluted to other aqueous solutions as needed . The reconstitution should be performed gently, allowing the lyophilized protein to dissolve completely before aliquoting or use. Avoid vigorous vortexing which may lead to protein denaturation.

What are the recommended storage conditions for Recombinant Mouse C10?

For long-term stability of Recombinant Mouse C10, adhere to these storage recommendations:

To maintain protein integrity, use a manual defrost freezer and avoid repeated freeze-thaw cycles . For working solutions, store in single-use aliquots to minimize freeze-thaw damage. The addition of carrier proteins (0.1% HSA or BSA) is recommended for dilute solutions to prevent adsorption to labware and maintain stability during storage .

How can Recombinant Mouse C10 be adapted for use in immunization studies?

To adapt Recombinant Mouse C10 for immunization studies, formulate the protein with appropriate adjuvants to enhance immune responses. Based on comparable immunization protocols, mix 100 μg of purified C10 protein with Freund's adjuvant in a 1:2 ratio for initial immunizations . For subsequent booster immunizations, consider using incomplete Freund's adjuvant or alternative adjuvants with lower reactogenicity.

The immunization schedule should include an initial immunization followed by booster doses at 2-3 week intervals. For subcutaneous administration, inject 100 μg/100 μl of protein on the back of the animal . To evaluate mixed protein effects, consider combining C10 with other immunogenic proteins (50 μg/50 μl of each protein) . Monitor both humoral immunity through antibody production and cellular immunity through cytokine profiling (particularly IL-4, IL-10, and IFN-γ) to evaluate Th1/Th2 balance in the immune response.

What methodological approaches can determine if Recombinant Mouse C10 activates specific immune cell populations?

To determine if Recombinant Mouse C10 activates specific immune cell populations, implement a multi-parameter approach combining cellular, molecular, and functional analyses:

• Flow cytometry analysis: Treat immune cell populations (neutrophils, macrophages, dendritic cells) with C10 protein and measure activation markers (CD69, CD86, MHC II) and intracellular signaling molecules (phosphorylated ERK, NFκB).

• Cytokine/chemokine profiling: Quantify secreted factors using multiplex ELISA or Luminex assays after stimulation with various concentrations of C10 (0.05-0.25 μg/ml) .

• Transcriptional analysis: Measure gene expression changes in stimulated cells using qPCR or RNA-seq, focusing on genes associated with immune activation and differentiation.

• Functional assays: Assess changes in functional capabilities including phagocytosis (for macrophages), antigen presentation, or respiratory burst (for neutrophils).

• Receptor blocking studies: Use CCR1 antagonists or antibodies to determine receptor-dependency of observed activation, as C10 has demonstrated interaction with the CCR1 receptor .

Include appropriate controls including unstimulated cells, cells treated with denatured protein, and cells treated with irrelevant proteins of similar size.

What are common challenges in measuring the biological activity of Recombinant Mouse C10?

Common challenges in measuring the biological activity of Recombinant Mouse C10 include:

• Receptor expression variability: The target cells must express adequate levels of CCR1 receptors for consistent chemotactic responses. Regular validation of receptor expression in BaF3 transfectants or other target cells is essential.

• Protein activity loss: C10 may lose activity due to improper handling, storage, or freeze-thaw cycles. Implement single-use aliquoting and validate protein activity with each new lot.

• Assay sensitivity issues: The effective dose for chemotactic activity is narrow (ED50 of 0.5-2 ng/mL) , requiring careful concentration optimization within the 0.05-0.25 μg/ml range .

• Background migration: High spontaneous migration can mask specific chemotactic effects. Optimize serum concentrations and pre-treatment conditions to minimize background.

• Reproducibility challenges: Cell migration assays show inherent variability. Use technical replicates (minimum of 3) and biological replicates with appropriate statistical analyses to address this variability.

How can researchers distinguish between specific and non-specific effects when working with Recombinant Mouse C10?

To distinguish between specific and non-specific effects when working with Recombinant Mouse C10, implement these methodological controls:

• Receptor antagonism: Use specific CCR1 receptor antagonists to block C10-mediated effects. If the effect persists despite receptor blockade, it suggests non-specific activity.

• Concentration-response curve: Establish a complete concentration-response relationship, as specific receptor-mediated effects typically show bell-shaped or sigmoidal dose-response curves with saturation at higher concentrations.

• Heat-inactivated controls: Compare native C10 with heat-denatured protein to distinguish between effects requiring the native protein structure versus those caused by contaminants or non-specific protein interactions.

• Receptor-negative cell controls: Test C10 on cells lacking CCR1 expression to establish baseline non-specific responses.

• Endotoxin testing: Verify that recombinant protein preparations have minimal endotoxin levels (<0.1 EU/μg), as E. coli-derived proteins may contain contaminating endotoxins that activate immune cells independently of the protein itself .

How does the activity of Recombinant Mouse C10 compare with other CC chemokines in functional assays?

When comparing the activity of Recombinant Mouse C10 with other CC chemokines in functional assays, several distinguishing characteristics emerge:

• Receptor specificity: Unlike many CC chemokines that interact with multiple receptors, C10 predominantly signals through CCR1, making it more selective in its cellular targets compared to promiscuous chemokines like CCL5 (RANTES).

• Potency in chemotaxis: C10 demonstrates high potency with an ED50 of 0.5-2 ng/mL in CCR1-transfected BaF3 cell chemotaxis assays . This potency range is comparable to other established CC chemokines, though direct comparative studies within the same experimental system would be needed for precise rankings.

• Species-specificity: C10 is rodent-specific with no direct human homolog, unlike many other chemokines that show cross-species conservation. This limits direct translational studies but makes it valuable for rodent-specific immune pathway research.

• Cell-type selectivity: The protein shows particular activity on myeloid lineage cells and CCR1-expressing cells, distinguishing it from chemokines that predominantly target lymphoid populations.

When interpreting comparative data, researchers should account for differences in assay systems, receptor expression levels, and species-specific variations in receptor binding affinities.

What criteria should be used to evaluate the purity and identity of Recombinant Mouse C10 preparations?

To evaluate the purity and identity of Recombinant Mouse C10 preparations, researchers should implement the following analytical criteria:

• Purity assessment:

  • Conduct SDS-PAGE analysis with Coomassie or silver staining, expecting >95% purity

  • Perform RP-HPLC analysis to detect potential contaminants or degradation products

  • Evaluate endotoxin levels using LAL assay (<0.1 EU/μg for research applications)

• Identity confirmation:

  • Verify molecular weight (approximately 11 kDa)

  • Confirm N-terminal sequence matching the expected sequence: GLIQEIEKED RRYNPPIIHQ...

  • Perform mass spectrometry to confirm protein mass and potential post-translational modifications

• Functional verification:

  • Validate biological activity through chemotaxis assay using CCR1-transfected BaF3 cells

  • Establish dose-response relationship within the expected ED50 range (0.5-2 ng/mL)

  • Confirm receptor specificity through receptor blocking studies

Commercial preparations typically meet these criteria with purity standards exceeding 95% as determined by both RP-HPLC and SDS-PAGE analyses . Researchers should request and review certificates of analysis for each lot to ensure consistency across experiments.

What novel applications of Recombinant Mouse C10 are emerging in immunological research?

Novel applications of Recombinant Mouse C10 in immunological research include:

• Vaccine adjuvant development: Similar to studies with other recombinant proteins like LpxC and GmhA , C10 could be investigated as an immune modulator or adjuvant that enhances specific immune responses. The ability to stimulate both Th1 and Th2 responses makes it potentially valuable for balanced immune activation.

• Myeloid cell differentiation studies: Given C10's expression in myeloid lineages and induction during myeloid differentiation , it could serve as a marker or modulator of myeloid cell development in bone marrow culture systems.

• Tissue-specific immune response modulation: As a chemokine that recruits specific immune cell populations, C10 could be used in targeted delivery systems to enhance localized immune responses in tissues of interest.

• Chimeric protein development: Fusion proteins combining C10 with antigens of interest could direct these antigens to CCR1-expressing antigen-presenting cells, potentially enhancing immune recognition and response.

• Immunotherapy combinations: C10 could be evaluated in combination with checkpoint inhibitors or other immunotherapeutics to enhance recruitment of effector cells to sites of interest.

These applications would require careful experimental design with appropriate controls and validation of immune response parameters through methods such as cytokine profiling (IL-4, IL-10, IFN-γ) to assess Th1/Th2 balance .

How might genetic modifications of Recombinant Mouse C10 alter its functional properties?

Genetic modifications of Recombinant Mouse C10 could significantly alter its functional properties in several ways:

• Receptor specificity alterations: Targeted mutations in the receptor-binding regions could modify the protein's affinity for CCR1 or potentially enable binding to additional chemokine receptors, expanding its cellular targets.

• Half-life extensions: Fusion with serum albumin binding domains or PEGylation sites could extend the protein's circulatory half-life, enabling prolonged activity in vivo without frequent dosing.

• Antagonist development: Strategic modifications could convert C10 from a receptor agonist to an antagonist, useful for blocking inflammatory responses mediated by the CCR1 receptor.

• Enhanced potency: Structure-guided mutations aimed at optimizing receptor interactions could potentially lower the effective concentration below the current ED50 of 0.5-2 ng/mL .

• Altered oligomerization properties: Modifications affecting the protein's tendency to form dimers or higher-order structures could change its functional presentation to cells and receptors.