Recombinant Mouse Pro-epidermal growth factor (Egf), partial (Active)

What is recombinant mouse EGF and what are its structural characteristics?

Recombinant mouse EGF (Epidermal Growth Factor) is a bioactive protein produced in expression systems (typically E. coli) that mimics the naturally occurring growth factor in mice. It stimulates the growth of various epidermal and epithelial tissues both in vivo and in vitro, as well as certain fibroblast populations in cell culture . The protein contains three intramolecular disulfide bonds that are critical for its proper folding and biological activity .

Structurally, mouse EGF exists in different forms depending on preparation methods:

• Molecular Weight: Reported as 6.2-7.2 kDa as a monomer

• Some preparations report it as a homodimer with MW of 12.4 kDa

• Length: Typically 54 amino acids

• Expression region: Often representing amino acids 977-1029 of the full pro-EGF sequence

• Purity: Commercial preparations typically exceed 95% purity as determined by SDS-PAGE analysis

How should recombinant mouse EGF be stored and reconstituted for optimal activity?

Proper storage and reconstitution are critical for maintaining the biological activity of recombinant mouse EGF. Based on manufacturer recommendations:

Storage recommendations:

• Lyophilized product should be stored at -20°C or -80°C

• Avoid repeated freeze-thaw cycles as they can compromise biological activity

• Working aliquots may be stored at 4°C for up to one week

Reconstitution protocol:

• Briefly centrifuge the vial before opening to bring contents to the bottom

• Reconstitute the lyophilized protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% for long-term storage (50% is commonly recommended)

• Prepare small aliquots to avoid repeated freeze-thaw cycles

• Store reconstituted aliquots at -20°C/-80°C for long-term storage

How is the biological activity of recombinant mouse EGF measured?

The biological activity of recombinant mouse EGF is typically assessed through cell-based proliferation assays. Two common methodological approaches include:

BALB/c 3T3 cell proliferation assay:

• Mouse EGF stimulates dose-dependent proliferation of BALB/c 3T3 cells

• The ED50 (effective dose for 50% maximal response) is typically less than 250 pg/ml

• This corresponds to an expected specific activity of approximately 4 × 10^6 units/mg

A431 cell proliferation inhibition assay:

• At higher concentrations, EGF can inhibit the proliferation of human epithelial A431 cells

• The ED50 for this inhibitory effect is typically 2-20 ng/ml

These standardized assays provide quantitative measures of biological activity and ensure lot-to-lot consistency in research applications.

What formulations are commonly available for recombinant mouse EGF?

Recombinant mouse EGF is typically provided as a lyophilized powder with various buffer compositions:

The low endotoxin levels (<1.0 EU/μg) are critical for in vivo applications and sensitive cell culture experiments to prevent non-specific inflammatory responses that could confound experimental results .

How does recombinant mouse EGF interact with the EGFR signaling pathway?

Recombinant mouse EGF binds with high affinity to the epidermal growth factor receptor (EGFR), a transmembrane tyrosine kinase receptor. This binding initiates a complex signaling cascade that includes:

• Receptor dimerization and autophosphorylation: Upon EGF binding, EGFR undergoes conformational changes leading to dimerization and autophosphorylation of tyrosine residues in the cytoplasmic domain

• Intracellular signal transduction: Phosphorylated EGFR recruits adaptor proteins and activates multiple downstream pathways including:

  • MAPK (Mitogen-Activated Protein Kinase) pathway

  • PI3K/Akt pathway

  • JAK/STAT pathway

  • PLCγ/PKC pathway

• Receptor trafficking and down-regulation: Following activation, EGFR undergoes endocytosis, which can lead to either recycling back to the cell surface or lysosomal degradation. This process is regulated by complex interactions involving the E3 ubiquitin ligase Cbl, the guanine exchange factor (GEF) Cool-1 (β-Pix), and the Rho family G protein Cdc42

• Magnesiotropic effects: EGF acts as a magnesiotropic hormone that stimulates magnesium reabsorption in the renal distal convoluted tubule through engagement of EGFR and activation of the magnesium channel TRPM6

The complexity of these signaling networks necessitates careful experimental design when studying specific aspects of EGF-EGFR interactions.

What role does the Cbl/Cool-1/Cdc42 complex play in EGF receptor signaling and how can it be studied?

The Cbl/Cool-1/Cdc42 protein complex represents an important regulatory mechanism in EGFR signaling that has been identified as critical in disrupting receptor down-regulation . Understanding this complex requires sophisticated experimental approaches:

Role of the complex:

• E3 ubiquitin ligase Cbl normally mediates ubiquitination of activated EGFR, targeting it for degradation

• The guanine exchange factor Cool-1 (β-Pix) can sequester Cbl into a complex with Cdc42

• Formation of this complex reduces Cbl-mediated ubiquitination of EGFR, thus inhibiting receptor down-regulation

• This mechanism may contribute to enhanced and prolonged EGFR signaling

Methodological approaches to study this complex:

• Differential equation modeling: Mathematical models can predict the dynamics of complex formation and its effects on EGFR trafficking and signaling

• Co-immunoprecipitation assays: To detect physical interactions between Cbl, Cool-1, and Cdc42

• siRNA knockdown experiments: To assess the functional consequences of reducing individual components

• Fluorescence microscopy: To visualize complex formation and localization in cells

• Optimal experimental design: Applying mathematical approaches to identify which experiments would most effectively reduce uncertainty in model predictions

These methods can be combined with recombinant mouse EGF stimulation to investigate how this complex regulates EGFR signaling dynamics in various cellular contexts.

How can mathematical modeling be applied to EGF receptor signaling studies?

Mathematical modeling provides powerful tools for understanding the complex dynamics of EGFR signaling. Differential equation models are particularly useful for capturing the temporal evolution of signaling networks:

Key aspects of mathematical modeling of EGFR signaling:

• Model construction: Developing a set of differential equations representing the biochemical reactions in the EGFR pathway, including:

  • Receptor-ligand binding kinetics

  • Receptor dimerization and phosphorylation

  • Activation of downstream signaling molecules

  • Receptor trafficking between cellular compartments

  • Feedback regulatory mechanisms

• Parameter estimation: Determining rate constants and initial concentrations based on experimental data through methods such as:

  • Bayesian parameter estimation

  • Maximum likelihood estimation

  • Sensitivity analysis to identify most influential parameters

• Uncertainty quantification: Calculating confidence intervals for model predictions to assess reliability

• Optimal experimental design: Using the model to suggest new experiments that would most effectively reduce uncertainty in specific predictions, particularly for components that are difficult to measure directly

• Model validation: Testing model predictions against independent experimental data not used in model construction

This approach has been successfully applied to understanding complex aspects of EGFR signaling, including the role of the Cbl/Cool-1/Cdc42 complex in receptor down-regulation .

What factors affect the reproducibility of experiments using recombinant mouse EGF?

Achieving reproducible results with recombinant mouse EGF requires careful attention to multiple experimental factors:

Critical factors affecting reproducibility:

• Protein quality and handling:

  • Proper reconstitution following manufacturer protocols

  • Avoiding repeated freeze-thaw cycles

  • Using appropriate storage conditions (-20°C/-80°C for stock, 4°C for working aliquots)

  • Verifying activity before experiments using standardized assays

• Experimental conditions:

  • Cell density and passage number in cell-based assays

  • Serum starvation conditions prior to EGF stimulation

  • Medium composition and presence of other growth factors

  • Duration and temperature of EGF treatment

• Detection methods:

  • Sensitivity and dynamic range of assays used to measure responses

  • Appropriate controls (positive, negative, vehicle)

  • Technical replicates to assess measurement variability

  • Biological replicates to assess biological variability

• Data analysis:

  • Appropriate statistical methods for the experimental design

  • Normalization procedures for comparing across experiments

  • Transparent reporting of all experimental parameters

• Lot-to-lot variability:

  • Different production lots may have slight variations in activity

  • ED50 values should be verified for each new lot

  • Using the same lot for all experiments within a study when possible

Careful documentation and standardization of these factors can significantly improve experimental reproducibility when working with recombinant mouse EGF.

What methodologies are recommended for studying EGF-induced cellular responses in different experimental models?

Various methodological approaches can be employed to study EGF-induced cellular responses, depending on the specific research question:

For studying proliferation responses:

• Cell counting: Direct enumeration using hemocytometer or automated cell counters

• MTT/MTS assays: Colorimetric assays measuring metabolic activity

• BrdU incorporation: Measuring DNA synthesis as an indicator of proliferation

• Ki-67 immunostaining: Detecting cells in active phases of the cell cycle

• BALB/c 3T3 cells: Commonly used model system with ED50 < 250 pg/ml

For studying receptor activation and signaling:

• Western blotting: Detecting phosphorylation of EGFR and downstream targets

• Phospho-specific flow cytometry: Measuring phosphorylation at single-cell resolution

• Proximity ligation assay: Detecting protein-protein interactions in situ

• Live-cell imaging: Monitoring receptor trafficking and signaling dynamics

• RNA-seq/microarray analysis: Measuring transcriptional responses to EGF stimulation

For studying receptor trafficking:

• Fluorescently-labeled EGF: Tracking ligand-receptor complexes

• Immunofluorescence microscopy: Visualizing receptor localization

• Surface biotinylation: Quantifying receptor internalization rates

• Sucrose gradient fractionation: Separating cellular compartments

• Differential equation modeling: Predicting trafficking dynamics based on experimental data

These methodologies can be combined to provide comprehensive insights into EGF-induced cellular responses across different experimental models.