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

What is the molecular structure of recombinant mouse TNF-alpha protein?

Recombinant mouse TNF-alpha is typically expressed as a partial protein spanning amino acids 80-235 of the native sequence, with an N-terminal methionine. The biologically active form exists as a homotrimer with a molecular weight of approximately 50.8 kDa as determined by SEC-MALS analysis . The protein consists of a 35 amino acid cytoplasmic domain, a 21 amino acid transmembrane segment, and a 179 amino acid extracellular domain (ECD). Within the extracellular domain, mouse TNF-alpha shares 94% amino acid sequence identity with rat and 70-77% with various other species including human TNF-alpha .

How should recombinant mouse TNF protein be reconstituted and stored for optimal stability?

For optimal stability, lyophilized recombinant mouse TNF should be reconstituted at a concentration of 100 μg/mL in sterile PBS containing at least 0.1% human or bovine serum albumin as carrier protein . After reconstitution:

• Aliquot into polypropylene microtubes to avoid repeated freeze-thaw cycles

• Store at -80°C for long-term stability

• Never dilute to concentrations below 50 μg/mL for long-term storage

• Use a manual defrost freezer to avoid temperature fluctuations

For carrier-free preparations, reconstitute in sterile PBS at the same concentration, but be aware that stability may be reduced without the carrier protein .

What is the biological activity of recombinant mouse TNF-alpha and how is it measured?

The biological activity of recombinant mouse TNF-alpha is commonly measured through its cytotoxic effect on the L-929 mouse fibroblast cell line in the presence of actinomycin D (a metabolic inhibitor). The effective dose that induces 50% cytotoxicity (ED50) ranges from 8-50 pg/mL, with this bioassay serving as a standard measure of potency .

Methodology for bioactivity testing:

• Culture L-929 cells to appropriate confluence

• Add actinomycin D to sensitize cells

• Treat with serial dilutions of recombinant mouse TNF-alpha

• Measure cell viability after 24 hours

• Calculate ED50 from dose-response curve

This standardized assay allows researchers to compare the relative potency of different preparations and ensure consistent biological activity across experiments.

How should recombinant mouse TNF-alpha be used in an ELISA system?

Recombinant mouse TNF-alpha serves as an effective quantitative standard in sandwich ELISA systems. For optimal results:

• Create a standard curve using serial dilutions of the recombinant protein

• Prepare fresh dilutions in sample diluent with 5-10 mg/mL carrier protein (BSA)

• Use purified capture antibody (e.g., G281-2626) coated on microwells

• Apply biotinylated detection antibody (e.g., MP6-XT3) following sample incubation

• Develop with streptavidin-HRP and appropriate substrate

• Calculate sample concentrations using the standard curve

The limit of detection for mouse TNF-alpha in a well-optimized ELISA is approximately 3.7 pg/mL . For reproducible results, maintain consistent incubation times and temperatures across assays.

What are the considerations for using recombinant mouse TNF-alpha in in vivo models?

When designing in vivo experiments with recombinant mouse TNF-alpha:

• Dosage and administration: Based on published research, effective dosing ranges vary by model. In airway inflammation models, extended administration (30 days) has demonstrated significant physiological effects .

• Expected outcomes: Recombinant TNF-alpha administration in mouse models has been shown to:

  • Increase airway resistance measured by whole-body plethysmography

  • Enhance inflammatory cell accumulation in airways

  • Upregulate mucus secretion from airway epithelium

  • Promote tissue remodeling through epithelial-mesenchymal transition (EMT)

• Duration considerations: Chronic exposure (30+ days) may be necessary to observe significant tissue remodeling effects, while acute responses can be detected within hours or days.

• Route selection: Select the administration route (intraperitoneal, intranasal, intratracheal) based on your target tissue and research question.

How can consistency be maintained when using recombinant mouse TNF-alpha across multiple experiments?

Maintaining experimental consistency requires:

• Standardization: Use the same source, lot number, and reconstitution protocol when possible

• Validation: Perform bioactivity testing (L-929 cytotoxicity assay) with each new lot

• Storage discipline: Create single-use aliquots to avoid freeze-thaw cycles

• Quality control: Verify protein concentration and purity (≥95% by SDS-PAGE is standard)

• Endotoxin monitoring: Ensure levels remain below 0.1 ng per μg of TNF-alpha to prevent confounding inflammatory responses

Table: Intra-assay reproducibility for mouse TNF-alpha detection

How does recombinant mouse TNF-alpha influence the TL1A/DR3 signaling axis in airway inflammation models?

Research has demonstrated that recombinant mouse TNF-alpha significantly impacts the TL1A/DR3 signaling axis in experimental asthma models. When administered to mice for 30 days:

• TNF-alpha upregulates both TL1A (TNF-like ligand 1A) and DR3 (Death Receptor 3) expression

• This upregulation correlates with increased markers of epithelial-mesenchymal transition (EMT)

• The effect is observable through increased expression of mesenchymal markers including collagen I, fibronectin, and vimentin

• Histological analysis reveals enhanced airway remodeling, collagen deposition, and α-SMA expression

Mechanistically, this suggests that chronic TNF-alpha exposure promotes airway remodeling through the TL1A/DR3 axis, identifying a potential therapeutic target for TNF-α-mediated inflammatory disorders. The pathway appears to be a key mediator between inflammatory cytokine signaling and tissue structural changes.

What are the methodological considerations for using recombinant mouse TNF-alpha in 3D culture systems versus traditional 2D cultures?

When transitioning from 2D to 3D culture systems with recombinant mouse TNF-alpha:

• Diffusion kinetics: Calculate adjusted concentrations accounting for reduced diffusion in 3D matrices. Typically, higher initial concentrations (1.5-2× higher) may be needed to achieve equivalent cellular exposure.

• Exposure duration: Extend treatment times to allow complete penetration through 3D structures. Monitor temporal response patterns to determine optimal treatment schedules.

• Analysis methods:

  • For 3D cultures, confocal microscopy with z-stack analysis is preferred over traditional plate-based assays

  • Consider using reporter cell lines embedded within 3D cultures to monitor real-time TNF signaling

  • Develop protocols for organoid or spheroid disaggregation that preserve protein and RNA integrity

• Validation approach: Always include 2D culture controls in parallel to establish baseline responses and comparative analyses.

• Data interpretation: Account for the microenvironmental differences when interpreting results, as 3D cultures often demonstrate altered receptor expression and signaling dynamics compared to 2D systems.

How can recombinant mouse TNF-alpha be used to study synergistic effects with other cytokines in immunological research?

To effectively study cytokine synergy with recombinant mouse TNF-alpha:

• Experimental design:

  • Use dose-response matrices rather than single combinations

  • Example: 5×5 matrix with 5 concentrations of TNF-alpha (0-100 ng/mL) against 5 concentrations of a second cytokine

  • Include appropriate single-cytokine controls at each concentration

• Analytical approaches:

  • Calculate synergy scores using established methods (Bliss independence, Loewe additivity, or ZIP models)

  • Perform temporal studies to distinguish between early and late synergistic effects

  • Examine both canonical (NF-κB, MAPK) and non-canonical pathways

• Readout selection:

  • Use multiple readouts spanning different biological processes (gene expression, protein phosphorylation, cellular phenotypes)

  • Consider high-dimensional approaches such as phospho-proteomics or single-cell RNA-seq to capture complex response patterns

• Statistical considerations:

  • Apply specialized statistical tools designed for synergy analysis

  • Account for inter-experimental variability through appropriate normalization strategies

What are the most common causes of reduced activity in recombinant mouse TNF-alpha preparations and how can they be addressed?

Common causes of reduced activity and their solutions include:

• Protein aggregation:

  • Problem: Multiple freeze-thaw cycles promote aggregation

  • Solution: Prepare single-use aliquots upon reconstitution

  • Verification: Check for visible precipitates or use dynamic light scattering

• Adsorption to surfaces:

  • Problem: Protein loss due to binding to container surfaces

  • Solution: Add carrier protein (0.1-1% BSA) to storage buffer and use low-binding tubes

  • Verification: Compare activity in different tube types

• Oxidation:

  • Problem: Reactive oxygen species damage functional epitopes

  • Solution: Include antioxidants in storage buffer and minimize air exposure

  • Verification: Mass spectrometry to detect oxidized residues

• Enzymatic degradation:

  • Problem: Contaminating proteases cleave active protein

  • Solution: Add protease inhibitors to stocks and working solutions

  • Verification: SDS-PAGE to detect degradation products

• Endotoxin contamination:

  • Problem: LPS contaminants confound biological responses

  • Solution: Use endotoxin-tested preparations (≤0.1 ng/μg protein)

  • Verification: LAL assay to measure endotoxin levels

How can researchers differentiate between direct effects of recombinant mouse TNF-alpha and secondary cellular responses in complex experimental systems?

To distinguish direct from secondary effects:

• Temporal analysis:

  • Track response kinetics with high temporal resolution (minutes to hours)

  • Direct effects typically occur within minutes to a few hours

  • Secondary effects emerge later (hours to days)

• Receptor blocking:

  • Use TNF receptor-specific blocking antibodies or soluble receptors

  • Compare responses in presence/absence of blockers

  • Direct effects should be completely abrogated by receptor blockade

• Transcriptional/translational inhibition:

  • Apply actinomycin D (transcription inhibitor) or cycloheximide (translation inhibitor)

  • Direct signaling effects persist while secondary responses requiring new gene expression are inhibited

  • Caution: These inhibitors have cytotoxic effects at longer timepoints

• Cell-specific knockouts/knockdowns:

  • Use conditional TNF receptor knockout models or cell-specific receptor knockdown

  • Allows mapping of direct responder cells versus secondary effector populations

• In silico network analysis:

  • Apply computational models to distinguish primary network perturbations from downstream cascades

  • Useful for complex multi-omics datasets where direct visualization of causality is challenging

What are the key considerations when comparing results obtained with different sources or preparations of recombinant mouse TNF-alpha?

When comparing results across different TNF-alpha preparations:

• Standardize by bioactivity rather than mass:

  • Use the L-929 cytotoxicity assay to normalize concentrations by biological activity (ED50)

  • Activity-matched concentrations provide more comparable results than mass-based dosing

• Account for structural differences:

  • Document exact amino acid sequence (full-length vs. partial)

  • Note the presence/absence of tags (His, GST, etc.)

  • Consider expression system differences (E. coli vs. mammalian)

• Formulation variables:

  • Carrier protein presence/absence affects stability and activity

  • Buffer composition influences protein behavior

  • Additives/preservatives may have biological effects

• Perform parallel validation:

  • When switching sources, run key experiments with both old and new preparations

  • Establish conversion factors for equivalent biological responses

  • Document lot-to-lot variation within the same supplier

• Reporting standards:

  • In publications, specify catalog number, lot number, and supplier

  • Report both mass concentration and biological activity

  • Describe storage and handling procedures in detail

By adhering to these methodological considerations, researchers can ensure more reproducible and comparable results when working with recombinant mouse TNF-alpha across different experimental systems.