Mouse basic Fibroblast Growth Factor Recombinant

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Mouse basic Fibroblast Growth Factor Recombinant

$70.00$1,300.00


accession P15655


Source Optimized DNA sequence encodingMousebasic Fibroblast Growth Factor mature chain was expressed in Escherichia Coli.
Molecular weight NativeMouseFGF basic isgenerated by the proteolytic removal of the signal peptide and propeptide,the molecule has a calulated mass of kDa. Recombinant mouse bFGFis a disulfide-linked homodimeric protein consisting of amino acid residue subunits, and migrates as an approximately16 kDa protein under non-reducing and reducing conditions in SDS-PAGE.
Purity >98%, as determined by SDS-PAGE and HPLC
Biological Activity The ED(50) was determined by the dose-dependent stimulation of thymidine uptake by BaF3 cells expressing FGF receptors is ≤.2 ng/ml, corresponding to a specific activity of ≥1 x units/mg.
Protein Sequence MAASGITSLP ALPEDGGAAF PPGHFKDPKR LYCKNGGFFL RIHPDGRVDG VREKSDPHVK LQLQAEERGV VSIKGVCANR YLAMKEDGRL LASKCVTEEC FFFERLESNN YNTYRSRKYS SWYVALKRTG QYKLGSKTGP GQKAILFLPM SAKS
Endotoxin Endotoxin content was assayed using a LAL gel clot method. Endotoxin level was found to be less than.1 ng/µg(1EU/µg).
Presentation Recombinant mouse basic FGF was lyophilized from.2 μm filtered PBS solution, pH.0.
Reconstitution A quick spin of the vial followed by reconstitution in distilled water to a concentration not less than.1 mg/mL. This solution can then be diluted into other buffers.
Storage The lyophilized protein is stable for at least years from date of receipt at -20° C. Upon reconstitution, this cytokine can be stored in working aliquots at° -° C for one month, or at -20° C for six months, with a carrier protein without detectable loss of activity. Avoid repeated freeze/thaw cycles.
Usage This cytokine product is for research purposes only.It may not be used for therapeutics or diagnostic purposes.


Biological Process Angiogenesis
Biological Process Differentiation
Molecular function Developmental-protein
Molecular function Growth-factor
Molecular function Heparin-binding

Methods

Neurosphere culture

  • Neurospheres were generated from cells isolated from the subventricular zone of 6–8 week old C57BL/6 mice and cultured in proliferation media consisting of DMEM/F12 (GibcoBRL/Invitrogen) containing 0.6% glucose, 3 mM NaHCO3, 5 mM HEPES, 2 mM L-glutamine, 0.1 mg/ml apo-transferrin, 25 µg/ml insulin, 60 µM putrescene, 30 mM sodium selenite, 20 nM progesterone and 1% BSA (all from), supplemented with EGF (20 ng/ml) and FGF2 (10 ng/ml), as previously described

Cell culture

  • The whole hearts extracted from male C57BL6 mice (12 weeks old) were perfused and washed several times with ice-cold PBS to remove the blood cells.
  • Aorta, pulmonary artery, and pericardium were removed carefully.
  • The hearts were minced and digested for 20-minutes at 37°C with 0.1% type-II collagenase and 0.01% DNAse-I .
  • The cells were passed through 40 µm filter, fractionated with 70%Percoll and cultured in maintenance medium containing serum-free DMEM/F12 supplemented with B27 , 20 ng/ml epidermal growth factor (EGF), and 40 ng/ml basic fibroblast growth factor (bFGF).
  • The cells were collected 1-week later and re-seeded on new culture dishes with serum-free maintenance medium at a low density (100cells/cm2) to initiate colony formation.
  • Sixteen days later, the colonies thus obtained were transferred into 24-well dish and cultured individually in expansion medium containing DMEM/F12 supplemented with 2%FBS, B27-Supplement , 20 ng/ml EGF , 40 ng/ml bFGF , and 10 ng/ml leukemia inhibitory…
  • The whole hearts extracted from male C57BL6 mice (12 weeks old) were perfused and washed several times with ice-cold PBS to remove the blood cells.
  • Aorta, pulmonary artery, and pericardium were removed carefully.
  • The hearts were minced and digested for 20-minutes at 37°C with 0.1% type-II collagenase and 0.01% DNAse-I .
  • The cells were passed through 40 µm filter, fractionated with 70%Percoll and cultured in maintenance medium containing serum-free DMEM/F12 supplemented with B27 , 20 ng/ml epidermal growth factor (EGF), and 40 ng/ml basic fibroblast growth factor (bFGF).
  • The cells were collected 1-week later and re-seeded on new culture dishes with serum-free maintenance medium at a low density (100cells/cm2) to initiate colony formation.
  • Sixteen days later, the colonies thus obtained were transferred into 24-well dish and cultured individually in expansion medium containing DMEM/F12 supplemented with 2%FBS, B27-Supplement , 20 ng/ml EGF , 40 ng/ml bFGF , and 10 ng/ml leukemia inhibitory factor (LIF).
  • All colony-derived cells were re-seeded on new dishes after they reached 90–100% confluence and were maintained with expansion medium.

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Cell culture

  • The whole hearts extracted from male C57BL6 mice (12 weeks old) were perfused and washed several times with ice-cold PBS to remove the blood cells.
  • Aorta, pulmonary artery, and pericardium were removed carefully.
  • The hearts were minced and digested for 20-minutes at 37°C with 0.1% type-II collagenase and 0.01% DNAse-I .
  • The cells were passed through 40 µm filter, fractionated with 70%Percoll and cultured in maintenance medium containing serum-free DMEM/F12 supplemented with B27 , 20 ng/ml epidermal growth factor (EGF), and 40 ng/ml basic fibroblast growth factor (bFGF).
  • The cells were collected 1-week later and re-seeded on new culture dishes with serum-free maintenance medium at a low density (100cells/cm2) to initiate colony formation.
  • Sixteen days later, the colonies thus obtained were transferred into 24-well dish and cultured individually in expansion medium containing DMEM/F12 supplemented with 2%FBS, B27-Supplement , 20 ng/ml EGF , 40 ng/ml bFGF , and 10 ng/ml leukemia inhibitory…
  • The whole hearts extracted from male C57BL6 mice (12 weeks old) were perfused and washed several times with ice-cold PBS to remove the blood cells.
  • Aorta, pulmonary artery, and pericardium were removed carefully.
  • The hearts were minced and digested for 20-minutes at 37°C with 0.1% type-II collagenase and 0.01% DNAse-I .
  • The cells were passed through 40 µm filter, fractionated with 70%Percoll and cultured in maintenance medium containing serum-free DMEM/F12 supplemented with B27 , 20 ng/ml epidermal growth factor (EGF), and 40 ng/ml basic fibroblast growth factor (bFGF).
  • The cells were collected 1-week later and re-seeded on new culture dishes with serum-free maintenance medium at a low density (100cells/cm2) to initiate colony formation.
  • Sixteen days later, the colonies thus obtained were transferred into 24-well dish and cultured individually in expansion medium containing DMEM/F12 supplemented with 2%FBS, B27-Supplement , 20 ng/ml EGF , 40 ng/ml bFGF , and 10 ng/ml leukemia inhibitory factor (LIF).
  • All colony-derived cells were re-seeded on new dishes after they reached 90–100% confluence and were maintained with expansion medium.

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Soluble amyloid precursor protein alpha (sAPPα) enhances proliferation in an EGF/bFGF-independent manner.

bFGF
  • (b) A neurosphere formation assay was performed without growth factors (EGF and bFGF), and NPCs were treated with an inactive inhibitor , GM6001 , or GM6001 + recombinant sAPPα (GM+rec sAPPα).