Recombinant Rat C-C motif chemokine 6 protein (Ccl6) (Active)

What are the key structural and biochemical properties of Recombinant Rat C-C motif chemokine 6 protein (Ccl6)?

Recombinant Rat C-C motif chemokine 6 protein (Ccl6) is a purified active protein with a theoretical molecular weight of approximately 10.4 kDa. The protein's expression region typically spans amino acids 22-115 of the full sequence, representing the mature protein without signal peptide. The protein is generally produced in E. coli expression systems as a tag-free preparation to maintain native structure and function .

When working with this protein, researchers should note that it belongs to the CC chemokine family, characterized by adjacent cysteine residues in its structure. Like other chemokines, its biological activity depends on its three-dimensional conformation maintained by disulfide bonds. While specific crystal structure data for rat Ccl6 is limited, chemokines typically share a similar fold with an N-terminal region, three-stranded β-sheet, and C-terminal α-helix.

What are the optimal storage and handling conditions for maintaining Recombinant Rat C-C motif chemokine 6 protein (Ccl6) activity?

For optimal stability and retention of biological activity, Recombinant Rat C-C motif chemokine 6 protein (Ccl6) should be stored at -20°C. It's crucial to avoid repeated freeze/thaw cycles as these can significantly compromise protein integrity and biological activity . Aliquoting the protein upon first thaw is recommended to minimize freeze/thaw events.

When reconstituting lyophilized protein, researchers should refer to the specific datasheet accompanying the product. Generally, reconstitution in sterile, buffered solutions (often PBS with 0.1% BSA) is recommended. After reconstitution, the protein typically remains viable for approximately 12 months when stored properly . For experiments requiring extended storage of reconstituted protein, consideration should be given to adding carrier proteins (e.g., 0.1-1% BSA) to prevent adhesion to storage container surfaces and maintain stability.

How can Recombinant Rat C-C motif chemokine 6 protein (Ccl6) be used in chemotaxis assays?

When designing chemotaxis assays with Recombinant Rat C-C motif chemokine 6 protein (Ccl6), researchers should implement transwell migration systems similar to those used for other chemokines. Following established methodologies from related chemokine research, a standard transwell assay can be employed to evaluate the chemotactic potential of Ccl6 .

The experimental protocol typically involves:

• Preparing target cells (such as immune cells expressing appropriate receptors) in serum-free medium

• Adding various concentrations of Recombinant Rat C-C motif chemokine 6 protein (Ccl6) (ranging from 1-100 ng/mL) to the lower chambers

• Adding cells to the upper chambers (typically 1-5×10^5 cells/well)

• Incubating for 2-4 hours at 37°C with 5% CO2

• Quantifying migrated cells by flow cytometry or microscopy

To validate specificity, parallel experiments using neutralizing antibodies against Ccl6 can be conducted to confirm that the observed migration is specifically induced by the chemokine, similar to validation approaches used for other chemokines like CXCL16 .

What are appropriate in vivo models for studying Recombinant Rat C-C motif chemokine 6 protein (Ccl6) function?

When designing in vivo studies with Recombinant Rat C-C motif chemokine 6 protein (Ccl6), researchers should consider inflammatory and autoimmune disease models in rats. Drawing from methodologies used with other chemokines, several approaches can be employed:

• Direct administration: Like studies with CXCL12, Recombinant Rat C-C motif chemokine 6 protein (Ccl6) can be administered via intrathecal or local injection to evaluate its effects on specific tissues . Researchers typically use osmotic pumps or repeated injections to maintain consistent levels.

• Viral vector-mediated overexpression: Similar to CXCL12 studies, adeno-associated viral (AAV) vectors can be engineered to express Ccl6 in specific tissues. This approach allows for targeted, sustained expression of the chemokine in tissues of interest .

• Experimental autoimmune models: Ccl6 can be studied in models such as experimental autoimmune encephalomyelitis (EAE), where Lewis rats are immunized with spinal cord homogenate mixed with complete Freund's adjuvant followed by pertussis toxin administration .

For quantifying in vivo effects, researchers should assess parameters including clinical scores, histopathological changes, and immune cell infiltration in relevant tissues.

How can researchers quantify Recombinant Rat C-C motif chemokine 6 protein (Ccl6) expression in tissue samples?

For precise quantification of Recombinant Rat C-C motif chemokine 6 protein (Ccl6) in tissue samples, researchers should employ a multi-method approach combining protein and mRNA analysis:

• Western blot analysis: Following protocols similar to those used for CXCL12 detection, tissue samples should be homogenized in RIPA buffer, sonicated, and centrifuged to obtain protein lysates. Protein concentration should be standardized (approximately 2 μg/μl) and 20-30 μg loaded per lane on 8-15% SDS-PAGE gels. After transfer to PVDF membranes, blocks with 5% skimmed milk or 3% BSA in TBST for 1 hour, followed by overnight incubation with anti-Ccl6 primary antibodies (1:1000 dilution) at 4°C .

• ELISA: Commercial ELISA kits for rat Ccl6 can provide quantitative measurements with high sensitivity. For tissue samples, standardized protein extraction protocols should be followed with careful attention to matrix effects.

• qRT-PCR: For mRNA quantification, tissue RNA should be extracted using standard methods (TRIzol or commercial kits), reverse transcribed, and analyzed using Ccl6-specific primers with appropriate housekeeping gene controls.

• Immunohistochemistry/immunofluorescence: For spatial localization, tissue sections should be processed and stained with anti-Ccl6 antibodies, with careful validation of antibody specificity through appropriate controls.

What methods are available for studying Recombinant Rat C-C motif chemokine 6 protein (Ccl6) receptor binding and signaling?

To investigate Recombinant Rat C-C motif chemokine 6 protein (Ccl6) receptor interactions and downstream signaling, researchers can adapt methodologies employed for other chemokines:

• Direct binding assays: Following protocols similar to those used for CXCL16-CXCR6 interaction studies, researchers can use fluorescently labeled or tagged Ccl6 (e.g., Ccl6-Fc fusion proteins) to quantify binding to receptor-expressing cells via flow cytometry . Competitive binding assays with unlabeled chemokine can help determine binding specificity and affinity.

• Calcium flux assays: Cells expressing putative Ccl6 receptors can be loaded with calcium-sensitive dyes (e.g., Fluo-4 AM) and stimulated with various concentrations of Recombinant Rat C-C motif chemokine 6 protein (Ccl6). Changes in intracellular calcium can be measured using flow cytometry or fluorescence plate readers, similar to assays used for identifying chemokine-receptor interactions such as 6Ckine-CXCR3 .

• Signaling pathway analysis: Western blot analysis of phosphorylated signaling molecules (e.g., ERK, Akt, p38 MAPK) in cells treated with Recombinant Rat C-C motif chemokine 6 protein (Ccl6) can provide insights into activated pathways. Time-course experiments (typically 0-60 minutes post-stimulation) and dose-response studies (1-100 ng/mL) should be performed.

• Receptor antagonist studies: Co-administration of specific receptor antagonists can help identify the primary receptors mediating Ccl6 effects, similar to the use of AMD3100 as a CXCR4 antagonist in CXCL12 studies .

How should dosage and timing be optimized when using Recombinant Rat C-C motif chemokine 6 protein (Ccl6) in experimental models?

When determining optimal dosage and timing for Recombinant Rat C-C motif chemokine 6 protein (Ccl6) administration, researchers should consider several factors:

• In vitro dose-response relationships: Initial experiments should establish dose-response curves using concentrations ranging from 1-100 ng/mL, similar to concentrations used for other chemokines like CXCL12 (10 ng/mL) in cell culture systems . EC50 values should be calculated to guide further experiments.

• In vivo dosing considerations: For in vivo administration, researchers should start with doses extrapolated from similar chemokines, typically 1-10 μg per animal depending on administration route and experimental model. For example, in intrathecal administration studies similar to those conducted with AMD3100, a dose of approximately 40 μg per rat may serve as a starting point .

• Timing considerations:

  • For acute response studies: Measurements at 30 minutes, 1, 2, 4, and 24 hours post-administration

  • For chronic studies: Administration schedules ranging from daily to every 2-3 days

  • For disease models: Administration relative to disease induction or progression (e.g., before immunization, at disease onset, or during peak disease) as was done in EAE models

• Route of administration: The biological activity of Recombinant Rat C-C motif chemokine 6 protein (Ccl6) may vary depending on the route of administration (intravenous, subcutaneous, intrathecal, etc.). Researchers should conduct pilot studies to determine the optimal route for their specific research questions.

What control conditions are essential when designing experiments with Recombinant Rat C-C motif chemokine 6 protein (Ccl6)?

Robust experimental design with Recombinant Rat C-C motif chemokine 6 protein (Ccl6) requires several key controls:

• Vehicle controls: Administration of the same buffer used for Ccl6 reconstitution and dilution to control for potential buffer effects.

• Heat-inactivated protein control: Heat-denatured Ccl6 to control for non-specific protein effects while maintaining the same protein concentration.

• Neutralizing antibody controls: Co-administration of specific anti-Ccl6 neutralizing antibodies to confirm observed effects are specifically due to Ccl6 activity, similar to approaches used with other chemokines .

• Receptor antagonist controls: Where receptors are known, specific receptor antagonists can be used to confirm receptor-mediated effects.

• Recombinant protein from different expression systems: Comparing E. coli-derived Ccl6 with mammalian-expressed protein to control for potential differences in post-translational modifications.

• Genetic controls: In transgenic or knockout models, appropriate wild-type, heterozygous, and homozygous littermates should be included.

• Timing controls: For time-course studies, multiple timepoints should be evaluated to distinguish between acute and chronic effects of Ccl6 administration.

What are common challenges in detecting biological activity of Recombinant Rat C-C motif chemokine 6 protein (Ccl6) and how can they be addressed?

Researchers frequently encounter several challenges when assessing Recombinant Rat C-C motif chemokine 6 protein (Ccl6) activity:

• Loss of protein activity: To address this issue, researchers should:

  • Minimize freeze/thaw cycles by preparing single-use aliquots

  • Include carrier proteins (0.1-1% BSA) in storage buffers

  • Verify protein integrity by SDS-PAGE before experiments

  • Store at recommended temperature (-20°C)

• Low sensitivity in detection assays: Researchers can:

  • Optimize antibody concentrations when using immunodetection methods

  • Include positive controls (known active chemokines) in functional assays

  • Concentrate samples when necessary using appropriate methods (e.g., ultrafiltration)

  • Consider using amplified detection systems for Western blotting or ELISA

• High background in chemotaxis assays: Solutions include:

  • Pre-starving cells in serum-free medium before assays

  • Using chemotaxis medium with reduced serum or serum alternatives

  • Carefully washing membrane filters between steps

  • Including negative control wells to establish baseline migration

• Variable receptor expression: Researchers should:

  • Verify receptor expression on target cells before experiments

  • Consider pre-treating cells with cytokines known to upregulate relevant receptors

  • Use positive control cells with confirmed receptor expression

  • Perform parallel receptor expression analysis alongside functional assays

How can researchers address inconsistent results in immunological assays using Recombinant Rat C-C motif chemokine 6 protein (Ccl6)?

When facing reproducibility challenges in Recombinant Rat C-C motif chemokine 6 protein (Ccl6) experiments, researchers should implement the following strategies:

• Standardize protein handling procedures:

  • Use consistent reconstitution protocols

  • Prepare master stock solutions to minimize variation between experiments

  • Validate protein activity in established assays before complex experiments

  • Document lot-to-lot variations and adjust concentrations accordingly

• Control for experimental variables:

  • Maintain consistent cell passage numbers for in vitro experiments

  • Standardize animal age, weight, and housing conditions for in vivo studies

  • Use the same reagent batches throughout a study when possible

  • Implement standard operating procedures (SOPs) for all experimental protocols

• Implement robust validation approaches:

  • Confirm specificity using multiple techniques (e.g., neutralizing antibodies, receptor antagonists)

  • Include dose-response curves in each experiment

  • Perform parallel assays with well-characterized chemokines as positive controls

  • Consider multiple readout methods to cross-validate observations

• Address technical variability:

  • Validate antibody specificity with appropriate controls

  • Calibrate equipment regularly

  • Implement rigorous normalization procedures for quantitative data

  • Consider the impact of circadian rhythms on in vivo experiments