Recombinant Mouse Interleukin-6 protein (Il6) (Active)

What is mouse IL-6 and what are its primary biological functions?

Mouse IL-6 is a multifunctional cytokine with a molecular weight of approximately 21.7-28 kDa that plays crucial roles in immunity, tissue regeneration, and metabolism. The protein consists of 187 amino acid residues (position 25-211, with an N-terminal Met in recombinant versions). IL-6 functions as a potent inducer of the acute phase response and contributes significantly to host defense during infection and tissue injury. It regulates immune responses through multiple pathways and participates in hematopoiesis . In the innate immune response, IL-6 is synthesized by myeloid cells such as macrophages and dendritic cells upon recognition of pathogens through toll-like receptors at infection or injury sites. In adaptive immunity, IL-6 is essential for B-cell differentiation into immunoglobulin-secreting cells and plays a major role in CD4+ T cell subset differentiation, particularly in the development of T follicular helper cells required for germinal center formation .

What is the difference between "classic signaling," "trans-signaling," and "cluster signaling" for IL-6?

These three distinct signaling modes represent different mechanisms through which IL-6 initiates cellular responses:

Classic signaling occurs when IL-6 binds to membrane-bound IL-6R, after which this complex associates with the signaling subunit IL6ST/gp130 to activate downstream pathways. This mode is limited to cells expressing the IL-6R, primarily hepatocytes, monocytes, and resting lymphocytes .

Trans-signaling involves IL-6 binding to soluble IL-6R (sIL-6R), and this complex then interacts with gp130 on cells. Since gp130 expression is ubiquitous while IL-6R expression is restricted, trans-signaling substantially expands the range of IL-6-responsive cells. This mechanism enables IL-6 to affect cells that do not express the membrane-bound IL-6R .

Cluster signaling represents a cell-to-cell communication mode where membrane-bound IL-6:IL-6R complexes on "transmitter cells" activate IL6ST receptors on neighboring "receiver cells." This mechanism allows for localized, contact-dependent IL-6 signaling between adjacent cells .

Each signaling mode contributes differently to IL-6's physiological and pathological roles, making them important targets for experimental manipulation and therapeutic intervention.

How should recombinant mouse IL-6 be stored to maintain optimal activity?

Proper storage is critical for maintaining IL-6 bioactivity. Upon initial thawing, recombinant mouse IL-6 should be aliquoted into polypropylene microtubes and frozen at -80°C for future use. This prevents repeated freeze-thaw cycles that degrade protein activity . Alternatively, the product can be diluted in sterile neutral buffer containing carrier protein (0.5-10 mg/mL), aliquoted, and stored at -80°C . For long-term storage, lyophilized preparations are more stable than solutions. Carrier-free formulations should not be frozen but stored according to manufacturer specifications, typically at 2-8°C . Research indicates that recombinant proteins stored with proper carrier proteins maintain activity significantly longer than those without, with studies showing >95% activity retention after 6 months when stored with BSA at -80°C compared to substantial activity loss within weeks when stored improperly.

What is the recommended reconstitution protocol for lyophilized mouse IL-6?

For optimal reconstitution of lyophilized mouse IL-6:

• Reconstitute at 100 μg/mL in sterile PBS containing at least 0.1% human or bovine serum albumin as a carrier protein

• Allow the protein to sit for 15-20 minutes at room temperature to fully dissolve

• Gently mix by pipetting or inverting the tube, avoiding vigorous vortexing which may denature the protein

• If using for in vitro biological assays, carrier protein concentrations of 0.5-1.0 mg/mL are recommended

• For use as an ELISA standard, higher carrier protein concentrations (5-10 mg/mL) are recommended

Failure to add carrier protein may result in significant activity loss due to protein adherence to tube walls and oxidative damage. Carrier proteins should be pre-screened for experimental compatibility as they may influence results through toxicity, endotoxin content, or blocking activity .

How can researchers verify the purity and activity of recombinant mouse IL-6?

Researchers should verify both purity and bioactivity of IL-6 preparations:

Purity Assessment:

• SDS-PAGE analysis under reducing conditions should show a single band at approximately 21-24 kDa

• Recombinant mouse IL-6 from reputable suppliers is typically ≥95% pure as determined by SDS-PAGE and absorbance assays based on the Beers-Lambert law

• Silver staining can provide enhanced sensitivity for detecting contaminants

Activity Validation:

• Bioactivity should be assessed through cell proliferation assays using the T1165.85.2.1 mouse plasmacytoma cell line, with expected ED50 values of 10-100 pg/mL

• Endotoxin levels should be ≤0.1 ng per μg of mouse IL-6 (or ≤1 EU/μg), as measured by chromogenic LAL assay, to avoid confounding experimental results

• Functional binding assays to IL-6R can provide additional confirmation of proper protein folding

Rigorous quality control ensures experimental reproducibility and prevents misleading results from inactive or contaminated preparations.

What are the key specifications to verify when purchasing recombinant mouse IL-6?

When selecting recombinant mouse IL-6 for research, verify these critical specifications:

Additionally, verify whether the preparation contains carrier proteins, as carrier-free formulations are preferred for certain applications where BSA might interfere . The presence of tags or fusion proteins should also be considered as these may affect activity or introduce experimental artifacts.

What are the optimal conditions for using mouse IL-6 in cell culture experiments?

For optimal results in cell culture experiments with mouse IL-6:

Concentration Range: The effective concentration varies by cell type and readout:

• For T cell differentiation assays: 10-50 ng/mL

• For B cell stimulation: 5-20 ng/mL

• For hepatocyte acute phase response induction: 1-10 ng/mL

• For proliferation assays with T1165.85.2.1 cells: starting at 100 pg/mL with expected ED50 of 10-100 pg/mL

Buffer Conditions:

• Physiological pH (7.2-7.4) in serum-free or low-serum medium

• Addition of 0.1-0.5% BSA to prevent non-specific binding and protein degradation

• Avoid media with high levels of IL-6 neutralizing factors

Timing Considerations:

• Acute responses: 0.5-6 hours post-stimulation

• Proliferative responses: 24-72 hours

• Differentiation experiments: 3-7 days with replenishment every 48-72 hours

Co-stimulation: IL-6 activity is often synergistically enhanced by co-treatment with soluble IL-6R for trans-signaling studies or with other cytokines (IL-21, IL-23) for specific T cell subset differentiation .

How should mouse IL-6 be used as a standard in ELISA development?

When using mouse IL-6 as an ELISA standard:

• Reconstitute lyophilized IL-6 in buffer containing 5-10 mg/mL carrier protein (typically BSA) to enhance stability and prevent loss through adsorption

• Create standard curves using 7-8 points of 2-fold serial dilutions, starting at 1000 pg/mL and extending to 7.8 pg/mL

• Include a zero standard (diluent only) for background determination

• Prepare fresh standards for each assay, or aliquot and store at -80°C, avoiding more than one freeze-thaw cycle

• Use purified MP5-20F3 as a capture antibody and biotinylated MP5-32C11 as a detection antibody for sandwich ELISA construction

• Validate the standard curve with recombinant IL-6 from multiple sources to ensure consistency

• Include reference samples with known IL-6 concentrations to monitor inter-assay variability

Calibrating standards against international reference preparations (when available) improves comparability between different laboratories and assay systems.

Why might there be loss of IL-6 bioactivity in experiments, and how can this be prevented?

Several factors can contribute to IL-6 activity loss:

Common Causes of Activity Loss:

• Repeated freeze-thaw cycles causing protein denaturation

• Improper storage temperature (room temperature exposure)

• Protein adsorption to container surfaces

• Proteolytic degradation

• Oxidative damage

• Inappropriate buffer conditions

Prevention Strategies:

• Aliquot IL-6 immediately after reconstitution to minimize freeze-thaw cycles

• Add carrier proteins (0.5-10 mg/mL BSA or HSA) to prevent adsorption and stabilize protein structure

• Store at -80°C in polypropylene tubes (not glass or polystyrene) to minimize adsorption

• Add protease inhibitors when working with complex biological samples

• Include reducing agents (DTT, β-mercaptoethanol) at low concentrations when appropriate

• Use sterile technique to prevent microbial contamination

• For long experimental timeframes, replenish IL-6 every 48-72 hours in cell culture

Implementing these measures can significantly improve experimental reproducibility and reduce variability in IL-6-dependent assays.

How can researchers troubleshoot inconsistent results in IL-6 bioactivity assays?

When facing inconsistent IL-6 bioactivity results:

Systematic Troubleshooting Approach:

• Verify reagent quality:

  • Check protein lot, expiration date, and storage conditions

  • Assess purity by SDS-PAGE under reducing conditions

  • Measure endotoxin levels that may confound results

• Examine cell culture conditions:

  • Verify responder cell passage number and culture conditions

  • Check for mycoplasma contamination

  • Ensure consistent cell density between experiments

  • Control for serum lot variability that may contain IL-6 inhibitors

• Review experimental protocol:

  • Standardize pipetting technique for consistent protein delivery

  • Calibrate equipment (incubators, plate readers)

  • Use positive controls (TNF-α, IL-1β) to verify cell responsiveness

  • Include reference standards for inter-assay normalization

• Analyze data handling:

  • Apply consistent curve-fitting methods for ED50 determination

  • Check for outliers and statistical approaches

  • Account for background signal appropriately

Addressing these factors systematically allows identification of variables affecting assay performance and implementation of appropriate controls for more reproducible results.

How can researchers effectively study IL-6 trans-signaling in experimental models?

Studying IL-6 trans-signaling requires specific experimental strategies:

Methodological Approaches:

• Designer Cytokine Fusion Proteins:

  • Use Hyper-IL-6 (fusion of IL-6 and sIL-6R) to specifically activate trans-signaling

  • Compare responses to equal concentrations of IL-6 alone to differentiate between classic and trans-signaling

• Selective Inhibition Strategies:

  • Apply sgp130Fc (soluble gp130 fusion protein) which specifically blocks trans-signaling without affecting classic signaling

  • Utilize neutralizing antibodies against IL-6 vs. antibodies against IL-6:sIL-6R complex

• Cell-Specific Approaches:

  • Use cells lacking membrane IL-6R but expressing gp130 to study pure trans-signaling

  • Perform co-culture experiments with IL-6R+ "transmitter" and reporter "receiver" cells for cluster signaling studies

• In vivo Models:

  • Compare IL-6 knockout, IL-6R conditional knockout, and gp130 conditional knockout mice

  • Utilize tissue-specific promoters to manipulate signaling components in target tissues

• Readout Systems:

  • Monitor STAT3 phosphorylation as a proximal signaling event

  • Measure trans-signaling-specific gene expression signatures

  • Track phenotypic outcomes unique to trans-signaling (e.g., specific T-cell differentiation patterns)

These approaches enable researchers to distinguish the contributions of different IL-6 signaling modes to biological processes and disease mechanisms.

What experimental designs can differentiate between pro-inflammatory and anti-inflammatory roles of IL-6?

IL-6 exhibits context-dependent pro- and anti-inflammatory functions that can be experimentally distinguished:

Experimental Approaches:

• Temporal Analysis:

  • Acute vs. chronic IL-6 exposure models

  • Time-course experiments tracking transitional inflammatory markers

  • Pulse-chase studies with labeled IL-6 to track signaling duration

• Cell-Specific Responses:

  • Compare responses in pro-inflammatory cells (Th17, macrophages) vs. regulatory cells (Tregs, M2 macrophages)

  • Use cell-type specific knockout or reporter systems

  • Examine tissue-resident vs. infiltrating immune cells in inflammatory models

• Signaling Pathway Discrimination:

  • Selectively inhibit JAK/STAT vs. MAPK/ERK pathways downstream of IL-6R

  • Compare classic vs. trans-signaling activation using Hyper-IL-6 and sgp130Fc

  • Examine differential gene expression profiles using RNA-seq or proteomics approaches

• Metabolic Context:

  • Study IL-6 effects during exercise vs. obesity models

  • Examine muscle-derived (myokine) IL-6 vs. adipose-derived IL-6

  • Investigate interactions with metabolic hormones and nutrients

• Disease Model Selection:

  • Compare IL-6 function in sterile inflammation vs. infectious models

  • Study acute injury healing vs. chronic inflammatory conditions

  • Examine regulatory mechanisms in autoimmune vs. tumor microenvironments

Through these experimental designs, researchers can untangle the complex, sometimes contradictory roles of IL-6 in different physiological and pathological contexts.

What are current approaches to studying IL-6 interactions with other cytokines in complex inflammatory networks?

Investigating IL-6 within cytokine networks requires sophisticated experimental designs:

Advanced Methodological Approaches:

• Multiplexed Cytokine Analysis:

  • Use cytokine bead arrays or multiplex ELISA to simultaneously measure multiple cytokines

  • Apply mass cytometry (CyTOF) to analyze up to 40 parameters per cell

  • Implement single-cell RNA-seq to identify cytokine-producing and -responding cell populations

• Systems Biology Techniques:

  • Construct mathematical models of cytokine network interactions

  • Use principal component analysis and other dimensional reduction techniques to identify cytokine signatures

  • Apply machine learning to identify patterns in complex cytokine data sets

• Co-stimulation Experiments:

  • Design factorial experiments with combinations of cytokines (IL-6, IL-1β, TNF-α, IL-21, etc.)

  • Implement sequential addition protocols to test priming effects

  • Use cytokine-blocking antibodies in combination to detect compensatory mechanisms

• Genetic Approaches:

  • Create compound cytokine or receptor knockout models

  • Use inducible systems for temporal control of cytokine expression

  • Apply CRISPR-Cas9 screens to identify novel regulators of cytokine cross-talk

• Spatial Analysis:

  • Implement multiplexed immunohistochemistry or in situ hybridization

  • Use tissue clearing techniques with 3D imaging for whole-organ cytokine mapping

  • Apply imaging mass cytometry for spatial resolution of multiple cytokines

These techniques enable researchers to move beyond reductionist approaches to understand IL-6 function within the complex inflammatory milieu that exists in vivo.

How are post-translational modifications of IL-6 being studied, and how do they affect function?

Post-translational modifications (PTMs) of IL-6 represent an emerging area of research with important functional implications:

Methodological Approaches for Studying IL-6 PTMs:

• Mass Spectrometry Techniques:

  • Use LC-MS/MS to identify and quantify specific modifications

  • Apply top-down proteomics to analyze intact IL-6 proteoforms

  • Implement phosphoproteomics, glycoproteomics, and other PTM-specific enrichment strategies

• Site-Directed Mutagenesis:

  • Generate IL-6 variants with modified PTM sites (e.g., N- or O-glycosylation site mutations)

  • Create phosphomimetic mutants (e.g., Ser→Asp) to simulate constitutive phosphorylation

  • Engineer IL-6 with altered ubiquitination sites to study degradation kinetics

• Recombinant Protein Engineering:

  • Compare E. coli-derived (non-glycosylated) vs. mammalian-expressed (glycosylated) IL-6

  • Use in vitro enzymatic modifications to create defined PTM patterns

  • Develop IL-6 bioconjugates with synthetic PTM mimetics

• Structural Biology Approaches:

  • Apply X-ray crystallography and cryo-EM to determine how PTMs affect IL-6:receptor interactions

  • Use hydrogen-deuterium exchange mass spectrometry to probe structural dynamics

  • Implement molecular dynamics simulations to predict PTM effects on protein flexibility

Current research indicates that while E. coli-expressed recombinant mouse IL-6 lacks glycosylation, it retains bioactivity in standard assays, suggesting these modifications primarily affect pharmacokinetics, stability, and immunogenicity rather than receptor binding and activation.

What are the latest techniques for cell-specific targeting of IL-6 signaling in disease models?

Advanced approaches for cell-specific manipulation of IL-6 signaling include:

Current Targeting Strategies:

• Genetic Approaches:

  • Cell-type specific Cre-loxP systems to delete IL-6, IL-6R, or gp130 in targeted populations

  • AAV-mediated gene delivery with cell-specific promoters

  • CRISPR-Cas9 base editing for introducing signaling-modifying mutations in specific cells

• Protein Engineering Technologies:

  • Bispecific antibodies targeting IL-6 or IL-6R plus cell-specific markers

  • Cell-targeted nanoparticles delivering IL-6 signaling modulators

  • Synthetic cytokine receptors with altered signaling properties and restricted expression

• Small Molecule Approaches:

  • Cell-permeable JAK/STAT inhibitors with targeted delivery systems

  • Proteolysis-targeting chimeras (PROTACs) for cell-specific degradation of signaling components

  • Photoswitchable or chemically-inducible inhibitors for spatiotemporal control

• Single-Cell Analysis Integration:

  • Pairing interventions with single-cell RNA-seq to identify responsive cell populations

  • Using CITE-seq to simultaneously profile surface proteins and transcriptomes after intervention

  • Spatial transcriptomics to map intervention effects in tissue context

These emerging techniques allow researchers to move beyond global IL-6 modulation to precisely control signaling in specific cell populations, potentially reducing off-target effects while enhancing therapeutic efficacy in disease models.

What are the key considerations for translating mouse IL-6 research findings to human systems?

When translating mouse IL-6 findings to human applications, researchers must consider:

These considerations form a framework for responsible translation of fundamental IL-6 biology from murine systems to human therapeutic development.

How is our understanding of IL-6 biology evolving, and what are promising future research directions?

The field of IL-6 research continues to evolve in several exciting directions:

• Single-Cell Resolution Analysis: Integration of single-cell technologies is revealing unprecedented heterogeneity in IL-6 production and responsiveness across cell populations. Future research will likely map complete cellular networks of IL-6 communication in health and disease.

• Structural Biology Advancements: Cryo-EM and other structural techniques are providing deeper insights into the conformational dynamics of IL-6:receptor complexes. These findings are informing the design of selective pathway modulators with improved specificity.

• Temporal Signaling Dynamics: New biosensor technologies allow real-time tracking of IL-6 signaling in living cells and tissues. Understanding signaling kinetics and oscillations may explain context-dependent outcomes of IL-6 stimulation.

• Microbiome Interactions: Emerging research suggests the gut microbiome significantly influences IL-6 production and signaling. Investigating these interactions may reveal novel therapeutic approaches targeting the microbiome-IL-6 axis.

• Precision Medicine Applications: Biomarker development is enabling identification of patients likely to benefit from IL-6-targeted therapies. Future research will refine these approaches for personalized intervention strategies.

• Novel Delivery Systems: Targeted delivery technologies are being developed to modulate IL-6 signaling in specific tissues while sparing beneficial IL-6 functions elsewhere. These approaches may overcome limitations of current systemic IL-6 inhibitors.