Recombinant Rat Fuctinin-2

What is Recombinant Rat FGF-2 and what are its primary biological functions?

Recombinant Rat FGF-2, also known as basic fibroblast growth factor (bFGF), is a 146 amino acid protein with a molecular weight of 16.4 kDa that functions as a monomer. It is primarily expressed by endothelial cells and serves as a critical mediator of angiogenesis (the formation of new blood vessels) . This growth factor demonstrates several important biological activities:

• Stimulation of endothelial cell proliferation and migration

• Cardioprotective functions during cardiac injury

• Maintenance of stem cells in an undifferentiated state

• Promotion of wound healing and tissue repair

• Regulation of developmental processes

The protein signals through fibroblast growth factor receptor (FGFR) tyrosine kinases after binding to heparin, initiating various downstream cellular responses .

What are the molecular characteristics of Recombinant Rat FGF-2?

Recombinant Rat FGF-2 has distinct molecular characteristics that are important for researchers to understand:

• Amino acid sequence: The full 146 amino acid sequence is: MPALPEDGGG AFPPGHFKDP KRLYCKNGGF FLRIHPDGRV DGVREKSDPH VKLQLQAEER GVVSIKGVCA NRYLAMKEDG RLLASKCVTE ECFFFERLES NNYNTYRSRK YSSWYVALKR TGQYKLGSKT GPGQKAILFL PMSAKS

• Tertiary structure: Contains β-trefoil structure typical of the FGF family

• Production: Typically expressed in E. coli expression systems

• Purity: Commercial preparations achieve ≥95% purity as determined by reducing and non-reducing SDS-PAGE

• Endotoxin levels: High-quality preparations contain ≤1.00 EU/μg as measured by kinetic LAL

These characteristics ensure consistent performance in experimental applications.

What are the optimal storage and handling conditions for Recombinant Rat FGF-2?

For maintaining maximum biological activity, researchers should follow these storage and handling protocols:

Storage recommendations:

• Store at -80°C in a manual defrost freezer

• Avoid repeated freeze-thaw cycles that can degrade protein activity

• For prolonged storage, dilute to working aliquots in a 0.1% BSA solution

Reconstitution procedure:

• Centrifuge vial before opening to ensure all material is at the bottom

• For lyophilized product, reconstitute with sterile water at 0.1 mg/ml

• Gently pipet and wash down the sides of the vial to ensure full recovery of the protein

• Allow several minutes for complete reconstitution

• DO NOT VORTEX, as this can denature the protein

Working solution preparation:

• After reconstitution, the protein can be further diluted into other aqueous solutions

• Consider preparing small working aliquots to minimize freeze-thaw cycles

• For dilute solutions, include carrier proteins (like BSA) to prevent adhesion to plasticware

Following these guidelines ensures optimal protein stability and experimental reproducibility.

How is the biological activity of Recombinant Rat FGF-2 assessed?

The biological activity of Recombinant Rat FGF-2 can be determined through several experimental approaches:

Standard proliferation assay:

• The activity is typically determined by the dose-dependent induced proliferation of NR6R 3T3 cells

• Effective concentrations are usually less than 1 ng/ml

• This corresponds to an expected specific activity of 1.0 x 10^6 units/mg

Comparative activity assessment methods:

Quality control considerations:

• Include positive controls (commercial FGF-2 with known activity)

• Use negative controls (heat-inactivated FGF-2)

• Test for heparin dependency by running parallel assays with and without heparin

• Verify protein purity using SDS-PAGE before activity testing

These methodologies allow for standardized assessment of FGF-2 activity across experimental conditions.

How is Recombinant Rat FGF-2 utilized in stem cell research?

Recombinant Rat FGF-2 is a critical component for human embryonic stem cell culture systems and is necessary for maintaining human embryonic stem cells in an undifferentiated state . The methodological application includes:

Concentration optimization:

• Typical working concentrations range from 4-10 ng/ml for maintaining pluripotency

• Higher concentrations (10-20 ng/ml) may be used during critical passages

• Each cell line may require specific optimization

Administration protocol:

• Add fresh FGF-2 to culture medium every 24 hours due to its short half-life at 37°C

• For feeder-free systems, supplement with higher concentrations (typically 8-10 ng/ml)

• For feeder-dependent systems, lower concentrations may be sufficient (4-5 ng/ml)

Experimental validation:

• Monitor pluripotency markers (Oct4, Nanog, Sox2) to confirm efficacy

• Include FGF-2 withdrawal controls to verify dependency

• Assess morphological changes and differentiation markers

By carefully optimizing these parameters, researchers can effectively use Recombinant Rat FGF-2 to maintain stem cell cultures in the desired undifferentiated state for extended periods.

What mechanisms underlie the angiogenic effects of Recombinant Rat FGF-2?

Recombinant Rat FGF-2 is a potent mediator of angiogenesis , promoting new blood vessel formation through multiple mechanisms:

Cellular effects:

• Direct stimulation of endothelial cell proliferation

• Enhancement of endothelial cell migration toward angiogenic stimuli

• Promotion of endothelial cell survival under stress conditions

• Organization of endothelial cells into tubular structures

Molecular pathways:

• Activation of MAPK/ERK signaling cascade driving proliferation

• Stimulation of PI3K/Akt pathway promoting survival

• Induction of matrix metalloproteinases facilitating extracellular matrix remodeling

• Cross-talk with VEGF signaling enhancing angiogenic response

Experimental models for studying FGF-2 angiogenesis:

Understanding these mechanisms provides insights for applications in tissue engineering, wound healing, and cardiovascular research.

How does Recombinant Rat FGF-2 exert cardioprotective effects?

Recombinant Rat FGF-2 has demonstrated significant cardioprotective functions during heart injury , operating through several key mechanisms:

Direct cytoprotective effects:

• Activation of pro-survival signaling pathways in cardiomyocytes

• Inhibition of cardiomyocyte apoptosis via Bcl-2 family protein regulation

• Protection against oxidative stress through antioxidant enzyme induction

• Preservation of mitochondrial function during ischemic stress

Tissue-level effects:

• Promotion of angiogenesis in ischemic myocardium

• Reduction of fibrotic scarring through matrix remodeling

• Recruitment of cardiac progenitor cells

• Modulation of inflammatory responses post-injury

Experimental approaches to study cardioprotection:

• In vitro cardiomyocyte survival assays under hypoxia/serum deprivation

• Ex vivo Langendorff heart preparations subjected to ischemia-reperfusion

• In vivo myocardial infarction models with various FGF-2 administration protocols

These cardioprotective properties make FGF-2 a valuable research target for developing therapeutic strategies for cardiac injury and heart failure.

What experimental considerations are important when investigating FGF-2's effects on cardiac tissue?

When studying the cardioprotective functions of Recombinant Rat FGF-2, several methodological considerations are critical:

Timing of administration:

Delivery methods:

• Direct intramyocardial injection for localized effect

• Intracoronary delivery for targeted myocardial exposure

• Systemic administration for ease of delivery but lower cardiac specificity

• Sustained release systems (hydrogels, nanoparticles) for prolonged exposure

Assessment parameters:

• Functional measurements: Echocardiography (LVEF, fractional shortening), pressure-volume relationships

• Tissue analysis: Infarct size quantification, TUNEL staining for apoptosis

• Molecular readouts: Western blotting for survival pathway activation (Akt, ERK)

• Vascular assessment: CD31 staining for capillary density, perfusion studies

Confounding variables to control:

• Age and sex of experimental animals

• Comorbid conditions (diabetes, hypertension)

• Timing and duration of follow-up assessments

• Batch validation of FGF-2 activity before experiments

Accounting for these methodological factors ensures rigorous investigation of FGF-2's cardioprotective properties.

How does Recombinant Rat FGF-2 interact with other growth factors in experimental systems?

In physiological and experimental settings, FGF-2 rarely acts in isolation. Understanding its interactions with other factors is essential:

Synergistic interactions:

• VEGF (Vascular Endothelial Growth Factor): FGF-2 and VEGF demonstrate potent synergy in promoting angiogenesis

• TGF-β (Transforming Growth Factor-β): Works with FGF-2 to maintain stem cell pluripotency

• IGF-1 (Insulin-like Growth Factor-1): Enhances FGF-2's proliferative effects on multiple cell types

Methodological approaches to study interactions:

• Combinatorial treatment experiments with factorial design

• Isobologram analysis to quantify synergy or antagonism

• Pathway analysis using phospho-protein arrays or RNA-sequencing

• Advanced 3D culture systems to better recapitulate tissue environments

Experimental design considerations:

• Include appropriate controls for each factor alone

• Consider the temporal sequence of factor addition

• Use concentrations that reflect physiological ranges

• Monitor multiple readouts to capture diverse responses

Understanding these complex interactions enhances our ability to manipulate cellular behavior in research applications and potential therapeutic development.

What are the key differences between endogenous rat FGF-2 and recombinant versions?

Understanding the distinctions between naturally occurring and recombinant FGF-2 is important for experimental design and interpretation:

Comparative characteristics:

Methodological implications:

• Recombinant FGF-2 provides consistent, reproducible results across experiments

• Endogenous sources may better represent physiological complexity

• For mechanistic studies, recombinant proteins offer clearer pathway analysis

• Validation studies comparing both sources are valuable for translational research

These differences highlight why standardized recombinant proteins are essential for controlled research, while understanding endogenous forms provides physiological context.