Recombinant Mouse Tumor necrosis factor (Tnf), partial (Active)

What is Recombinant Mouse TNF and what are its key structural features?

Recombinant Mouse Tumor Necrosis Factor (TNF) is a cytokine that exists as a homotrimer in solution. The full-length protein spans amino acids 57-235 when expressed in systems like HEK293 cells, while the partial active form typically encompasses residues 89-235 expressed in E. coli systems . The protein has a molecular weight of approximately 16.4 kDa in its partial form . The amino acid sequence of the expressed region in the partial active form is: DKPVAHVVANHQVEEQLEWLSQRANALLANGMDLKDNQLVVPADGLYLVYSQVLFKGQGCPDYVLLTHTVSRFAISYQEKVNLLSAVKSPCPKDTPEGAELKPWYEPIYLGGVFQLEKGDQLSAEVNLPKYLDFAESGQVYFGVIAL . This specific sequence configuration maintains the protein's key binding domains for receptor interaction while optimizing expression efficiency.

How do partial and full-length Recombinant Mouse TNF proteins differ in research applications?

The partial active form (typically amino acids 89-235) and full-length form (typically amino acids 57-235) of Recombinant Mouse TNF exhibit distinct properties that influence their research applications. The partial form expressed in E. coli systems often demonstrates robust activity in cytotoxicity assays, with an ED50 of less than 0.08 ng/ml when tested with L-929 mouse fibroblast cells in the presence of actinomycin D . The full-length form expressed in mammalian systems like HEK293 contains additional N-terminal sequences that may affect receptor binding dynamics but provides post-translational modifications more similar to native TNF .

What methods are used to verify the purity and activity of Recombinant Mouse TNF?

Multiple analytical approaches are required to comprehensively characterize Recombinant Mouse TNF preparations:

• Purity assessment: SDS-PAGE analysis typically demonstrates ≥95% purity for research-grade preparations . Proteins should appear as a single band at approximately 16.4 kDa for partial forms or slightly higher for full-length forms.

• Endotoxin testing: LAL (Limulus Amebocyte Lysate) testing is essential, with acceptable levels below 1.0 EU/μg for research applications and ideally <0.005 EU/μg for sensitive cell culture systems .

• Bioactivity determination: The definitive test remains the L-929 fibroblast cytotoxicity assay in the presence of actinomycin D, where biological activity is expressed as ED50 values. For partial active forms, expected values are typically <0.08 ng/ml .

• Mass spectrometry: This technique confirms the exact molecular mass and can verify sequence integrity through peptide mapping.

• Receptor binding assays: Direct binding to recombinant TNFRSF1A/TNFR1 and TNFRSF1B/TNFBR receptors can provide quantitative affinity measurements.

Researchers should verify that suppliers provide comprehensive certificates of analysis covering these parameters before proceeding with experiments.

What are the optimal conditions for using Recombinant Mouse TNF in cell-based assays?

When designing cell-based assays with Recombinant Mouse TNF, several critical parameters must be optimized:

Cell Selection and Preparation:

• L-929 mouse fibroblasts represent the gold standard for cytotoxicity assays but require pre-sensitization with actinomycin D (typically 1 μg/ml) .

• For immunological studies, primary macrophages, dendritic cells, or established macrophage cell lines (RAW264.7) provide physiologically relevant responses.

• Cell density should be optimized (typically 2-5×10⁴ cells/well in 96-well format) to ensure consistent responses.

TNF Concentration Range:

• Initial experiments should include a broad concentration range (0.01-100 ng/ml) to establish dose-response relationships.

• For cytotoxicity assays, concentrations producing 20-80% maximum response (typically 0.05-5 ng/ml) provide the most reliable quantitative data .

Incubation Conditions:

• Temperature stability is critical; standard conditions of 37°C with 5% CO₂ should be strictly maintained.

• Time course studies should be performed, as different cellular responses occur at different timepoints:

  • Early gene expression: 1-4 hours

  • Cytokine production: 6-24 hours

  • Apoptosis/cytotoxicity: 16-48 hours

Medium Selection:

• Serum can contain TNF-binding proteins that reduce effective concentration; consider reduced serum or serum-free conditions during the treatment period.

• Avoid repeated freeze-thaw cycles of the protein and maintain working aliquots at 4°C for up to one week .

How can researchers accurately measure TNF activity in biological samples?

Accurate measurement of TNF in biological samples requires selecting appropriate methodologies based on the experimental context:

ELISA Approaches:
The sandwich ELISA format provides high specificity and sensitivity for mouse TNF quantification:

• Commercial mouse TNF ELISA kits typically provide detection ranges of 15.6-1000 pg/ml with sensitivity around 3.9 pg/ml .

• Sample volume requirements range from 50-100 μl with assay completion in 1-5 hours .

• Matrix effects must be considered: serum, plasma, cell culture supernatants, and tissue homogenates may require different dilution protocols.

Bioactivity Assays:
For functional TNF measurement, the L-929 cytotoxicity assay remains the reference method:

• Typically performed with actinomycin D (1 μg/ml) sensitization.

• Activity is expressed as units/ml, where one unit causes 50% cytotoxicity.

• Standard curves using recombinant TNF with known activity (e.g., ED50 <0.08 ng/ml) are essential for calibration .

Western Blotting:
For detection of membrane-bound versus soluble forms:

• Specific antibodies recognizing distinct epitopes can differentiate between the 26 kDa membrane-bound precursor and the 17 kDa soluble form.

• Activation-induced cleavage can be monitored using this approach.

Sample preparation protocols must be validated for each biological material, with particular attention to protease inhibitors for tissue homogenates to prevent artificial degradation of TNF.

How does mouse TNF signaling differ from human TNF in comparative studies?

Understanding species-specific differences is crucial when extrapolating mouse studies to human applications:

Receptor Binding Specificity:

• Mouse TNF binds with high affinity to both mouse TNFRSF1A/TNFR1 and TNFRSF1B/TNFBR .

• Critical interspecies difference: mouse TNF binds efficiently to mouse TNF receptors but demonstrates limited activity on human cells, whereas human TNF activates both human and mouse receptors.

• This asymmetric cross-reactivity must be considered when designing xenograft or humanized mouse models.

Downstream Signaling Divergence:

• While core apoptotic and inflammatory pathways are conserved, quantitative differences exist in:

  • Kinetics of NF-κB activation

  • Pattern and timing of cytokine induction

  • Susceptibility to apoptosis versus necroptosis

Experimental Design Implications:

• For mechanistic studies on human cells using mouse models, humanized TNF mice or human TNF administration may be required.

• When comparing mouse and human systems, matched concentrations by biological activity rather than protein mass should be used.

• Researchers should validate key findings across species barriers before making translational claims.

This interspecies variation explains why many TNF-targeted therapeutics show promising results in mouse models but fail in human clinical trials.

What approaches are recommended for studying TNF's role in metabolism and insulin resistance?

TNF plays significant roles in metabolic regulation, particularly in insulin resistance development:

Experimental Models:

• In vitro: 3T3-L1 adipocytes, C2C12 myocytes, and primary mouse hepatocytes treated with 1-10 ng/ml TNF for 24-72 hours demonstrate insulin resistance.

• Ex vivo: Adipose tissue explants from lean vs. obese mice show differential TNF production and insulin sensitivity.

• In vivo: High-fat diet-induced obesity models with TNF knockout or TNF receptor knockout mice provide systemic insights.

Molecular Mechanisms to Analyze:

• TNF induces insulin resistance through multiple pathways:

  • Inhibition of insulin-induced IRS1 tyrosine phosphorylation

  • Induction of GKAP42 protein degradation in adipocytes

  • Interference with insulin receptor signaling through serine phosphorylation of IRS proteins

Assay Approaches:

• Glucose uptake assays: Measure 2-deoxyglucose uptake following insulin challenge in TNF-treated vs. control cells.

• Protein phosphorylation analysis: Western blotting for phospho-IRS1, phospho-Akt, and phospho-GSK3β reveals TNF's impact on insulin signaling.

• Lipolysis measurement: Glycerol release assays quantify TNF's effect on adipocyte lipid metabolism.

Experimental Design Considerations:

• Time-dependent effects are critical: acute (minutes to hours) vs. chronic (days to weeks) TNF exposure produces different metabolic outcomes.

• Concentration matters: physiological (0.1-1 ng/ml) vs. pathological (5-50 ng/ml) concentrations activate different signaling pathways.

How can researchers investigate TNF's role in osteoclastogenesis and bone remodeling?

TNF is a crucial mediator of bone resorption through its effects on osteoclast formation and function:

Experimental Systems:

• In vitro osteoclastogenesis assays:

  • Bone marrow-derived macrophages (BMDMs) cultured with RANKL (50-100 ng/ml) ± TNF (1-10 ng/ml).

  • RAW264.7 cells stimulated with RANKL ± TNF provide a more homogeneous alternative.

  • Readouts include TRAP staining for multinucleated osteoclast formation and pit formation assays on dentine slices.

• Ex vivo bone organ cultures:

  • Calvarial or long bone explants cultured with TNF demonstrate integrated tissue responses.

  • μCT analysis before and after treatment quantifies bone loss.

• In vivo models:

  • Supracalvarial TNF injection models acute inflammatory bone loss.

  • TNF transgenic mice or TNF receptor knockout models in arthritis settings reveal chronic effects.

Molecular Pathways to Investigate:
TNF promotes osteoclastogenesis through multiple mechanisms :

• Synergistic enhancement of RANKL-induced osteoclast formation

• Induction of M-CSF production by stromal cells

• Upregulation of RANK expression on osteoclast precursors

• Activation of NF-κB, AP-1, and NFATc1 transcription factors

Recommended Analytical Approaches:

• Gene expression analysis focusing on osteoclast differentiation markers (TRAP, cathepsin K, MMP9)

• Signaling analysis through phosphorylation status of key mediators (p38, JNK, ERK, NF-κB)

• Quantitative bone histomorphometry in animal models

How can researchers address variable cell responses to Recombinant Mouse TNF stimulation?

Inconsistent cellular responses to TNF stimulation represent a common challenge that can be systematically addressed:

Common Causes and Solutions:

• Variable TNF receptor expression:

  • Problem: Receptor levels fluctuate with cell passage and culture conditions.

  • Solution: Regularly quantify TNFR1/TNFR2 expression by flow cytometry or western blot; standardize passage numbers for experiments.

• Endotoxin contamination:

  • Problem: Even low levels of LPS (<0.1 EU/ml) can synergize with TNF.

  • Solution: Use TNF preparations with verified low endotoxin (<0.005 EU/μg) ; include polymyxin B controls in experiments.

• Protein degradation:

  • Problem: TNF activity diminishes with repeated freeze-thaw cycles.

  • Solution: Prepare small single-use aliquots; avoid storing working solutions for more than one week at 4°C .

• Cell density variations:

  • Problem: TNF responses are influenced by cell-cell contact and paracrine factors.

  • Solution: Standardize seeding density; use hemocytometer counting rather than approximations.

• Serum interference:

  • Problem: Serum contains TNF-binding proteins and TNF-modulating factors.

  • Solution: Use serum-free medium during TNF treatment or standardize serum lots.

Experimental Controls to Implement:

• Positive biological control: Include a cell line with well-characterized TNF response (e.g., L-929) in parallel.

• Activity verification: Perform L-929 cytotoxicity assay with each new TNF lot or after extended storage.

• Receptor blockade control: Include TNF receptor neutralizing antibodies to confirm specificity.

• Concentration curve: Always run a concentration series rather than a single TNF dose.

What are the common pitfalls when measuring TNF activity and how can they be avoided?

Accurate measurement of TNF activity requires careful attention to several potential sources of error:

ELISA-Based Measurement Issues:

• Hook effect at high concentrations:

  • Problem: Very high TNF levels can produce falsely low readings.

  • Solution: Test multiple sample dilutions; verify linearity across the dilution series.

• Matrix interference:

  • Problem: Different biological samples contain substances that affect antibody binding.

  • Solution: Prepare standard curves in the same matrix as samples; use sample-specific validation.

• Cross-reactivity:

  • Problem: Some antibodies cross-react with other TNF family members.

  • Solution: Verify antibody specificity using knockout control samples or recombinant protein panels.

Detection ranges for mouse TNF ELISA typically span 15.6-1000 pg/ml with sensitivity around 3.9 pg/ml .

Bioactivity Assay Challenges:

• Variable cell sensitivity:

  • Problem: L-929 cells show passage-dependent TNF sensitivity.

  • Solution: Include internal standard curve with each assay; maintain master cell banks.

• Actinomycin D variability:

  • Problem: Actinomycin D potency affects TNF cytotoxicity readout.

  • Solution: Titrate actinomycin D (0.5-2 μg/ml) with each new lot; standardize pre-incubation time.

• Readout method biases:

  • Problem: Different cytotoxicity detection methods (MTT, LDH release, neutral red) have varying sensitivities.

  • Solution: Select one consistent method; validate with multiple TNF concentrations.

How is Recombinant Mouse TNF being utilized in cancer immunotherapy research models?

TNF's dual roles in tumor biology—both pro-tumorigenic and anti-tumorigenic—make it a fascinating target in cancer immunotherapy research:

Current Research Applications:

• Tumor microenvironment modulation:

  • TNF administration can alter the immunosuppressive tumor microenvironment through:

    • Increased tumor vessel permeability enhancing immune cell infiltration

    • Upregulation of adhesion molecules on endothelial cells

    • Dendritic cell maturation and antigen presentation enhancement

  • Experimental approach: Intratumoral TNF injection (50-500 ng) in established tumor models followed by immune cell phenotyping.

• Combination therapy models:

  • TNF synergizes with checkpoint inhibitors (anti-PD-1, anti-CTLA-4) in multiple tumor models.

  • Key readouts include tumor regression kinetics, tumor-infiltrating lymphocyte analysis, and long-term survival.

• TNF-engineered cell therapies:

  • CAR-T cells engineered to produce local TNF upon tumor antigen recognition show enhanced efficacy.

  • Membrane-bound vs. secreted TNF formats result in different safety and efficacy profiles.

Experimental Design Considerations:

• Timing and dosing:

  • Low-dose TNF (0.1-1 μg) promotes immune activation while high-dose (5-10 μg) can induce tumor vessel collapse.

  • Pulsed vs. continuous exposure produces different outcomes.

• Delivery methods:

  • Systemic administration is limited by toxicity.

  • Site-specific delivery using nanoparticles, tumor-targeting antibodies, or local injection provides better therapeutic window.

• Genetic approaches:

  • TNF and TNFR knockout mice crossed with spontaneous tumor models reveal context-dependent roles.

  • Conditional TNF expression systems allow temporal control of TNF effects.

The dual capacity of TNF to induce tumor cell death directly and to stimulate anti-tumor immune responses when properly administered makes it a valuable tool in immuno-oncology research .

How can Recombinant Mouse TNF be applied in angiogenesis and vascular biology research?

TNF exhibits complex effects on vascular biology, making it an important factor in angiogenesis research:

Experimental Models:

• In vitro endothelial models:

  • Mouse endothelial cell lines (e.g., bEnd.3) or primary endothelial cells cultured with TNF (0.1-10 ng/ml).

  • Readouts include tube formation assays, migration assays, and analysis of adhesion molecule expression.

  • TNF induces VEGF production synergistically with IL1B and IL6, forming a pro-angiogenic cytokine network .

• Ex vivo assays:

  • Aortic ring assays treated with TNF ± VEGF demonstrate vessel sprouting responses.

  • Retinal explants reveal developmental angiogenesis patterns in response to TNF.

• In vivo models:

  • Matrigel plug assays with embedded TNF (10-100 ng/ml) assess angiogenic potential.

  • Corneal pocket assays provide a transparent tissue for visualizing TNF-induced neovascularization.

  • Developmental angiogenesis in TNF or TNFR knockout embryos reveals physiological roles.

Key Research Questions and Methodologies:

• Dual effects analysis:

  • At low concentrations (0.1-1 ng/ml), TNF promotes angiogenesis through VEGF induction.

  • At high concentrations (>10 ng/ml), TNF can inhibit endothelial proliferation and induce apoptosis.

  • Compare dose-response curves across multiple endothelial cell types to characterize this biphasic response.

• Receptor-specific effects:

  • TNFR1 signaling generally mediates cytotoxic responses.

  • TNFR2 signaling often promotes survival and proliferation.

  • Use receptor-specific neutralizing antibodies or cells from receptor knockout mice to dissect these pathways.

• Inflammatory angiogenesis:

  • TNF upregulates adhesion molecules (ICAM-1, VCAM-1, E-selectin) on endothelial cells.

  • This promotes leukocyte-endothelial interactions critical for inflammatory angiogenesis.

  • Quantify adhesion molecule expression by flow cytometry or immunohistochemistry after TNF treatment.

Understanding TNF's context-dependent roles in angiogenesis has significant implications for developing therapies targeting pathological neovascularization in cancer, inflammatory diseases, and retinopathies.

What are the considerations for studying TNF's intracellular domain (ICD) in dendritic cell biology?

Recent research has revealed that the TNF intracellular domain (ICD) plays important signaling roles beyond the conventional understanding of TNF as a secreted cytokine:

Experimental Approaches:

• ICD isolation and detection:

  • The TNF ICD form induces IL12 production in dendritic cells .

  • Detection requires specific antibodies recognizing the cytoplasmic domain rather than the extracellular domain.

  • Western blotting with subcellular fractionation can identify nuclear translocation of TNF ICD.

• Dendritic cell models:

  • Bone marrow-derived dendritic cells (BMDCs) from wild-type vs. TNF knockout mice reconstituted with full-length or ICD-truncated TNF.

  • Primary human monocyte-derived dendritic cells transfected with TNF constructs.

  • Analysis of maturation markers (CD80, CD86, MHC II) and cytokine production (especially IL-12).

• Reverse signaling investigation:

  • TNF can function as a receptor as well as a ligand (reverse signaling).

  • Stimulation with soluble TNFR1/2 or agonist antibodies against membrane TNF triggers this reverse signaling.

  • Measure downstream phosphorylation events and gene expression changes.

Technical Challenges and Solutions:

• Distinguishing conventional from reverse signaling:

  • Problem: Both pathways often operate simultaneously.

  • Solution: Use TNF receptor knockout systems; employ membrane-restricted TNF mutants.

• ICD detection sensitivity:

  • Problem: Low abundance of cleaved ICD forms.

  • Solution: Use proteasome inhibitors to prevent degradation; employ immunoprecipitation before western blotting.

• Temporal dynamics:

  • Problem: ICD generation and signaling are often transient.

  • Solution: Conduct detailed time-course experiments; use live-cell imaging with fluorescently tagged TNF constructs.

This emerging area of TNF biology suggests that TNF functions not only as a secreted cytokine but also as a bidirectional signaling molecule, with important implications for understanding dendritic cell biology and adaptive immune responses.