Neural Differentiation of Pluripotent Stem Cells

R&D Systems Recombinant Human FGF basic (146 aa) FGF basic is a member of the FGF family of at least 23 related mitogenic proteins which show 35 - 60% amino acid conservation. FGF acidic and basic, unlike the other members of the family, lack signal peptides and are apparently secreted by mechanisms other than the classical protein secretion pathway. Specifications and Use of R&D FGF basic Source of R&D FGF • A DNA sequence encoding the naturally occurring, N-terminally truncated, 146 amino acid residue isoform of human FGF basic (Pro 143 - Ser 288; NP_001997) (Abraham, J.A. et al., 1986, EMBO J. 5:2523 - 2528) was fused to an E. coli signal peptide. The protein was expressed in E. coli. • Note: This is one of multiple forms available for this protein. Check R&D Systems' website, www.RnDSystems.com, for a complete listing of the variants. R&D FGF Molecular Mass • The recoombinant FGF basic contains an extra N-terminal amino acid Ala from the signal peptide and has a predicted molecular mass of approximately 16.0 kDa. R&D FGF basic Purity• > 97%, as determined by SDS-PAGE and visualized by silver stain. Endotoxin Level • < 1.0 EU per 1 μg of the cytokine as determined by the LAL method. Activity of R&D FGF • Measured by its ability to stimulate proliferation of NR6R-3T3 fibroblasts (Raines, E.W. et al., 1985, Methods Enzym. 109:749).  • The ED50 for this effect is typically 0.1 - 0.6 ng/mL in a fluorometric assay using the redox sensitive dye, Resazurin (R&D Systems, Catalog # AR002). R&D FGF Formulation • Lyophilized from a 0.2 μm filtered solution in 20 mM Tris, 0.01 mM DTT, pH 7.0, containing 50 μg of bovine serum albumin per 1 μg of cytokine. Reconstitution of R&D FGF • It is recommended that sterile phosphate-buffered saline containing at least 0.1% human serum albumin or bovine serum albumin and 1mM DTT be added to the vial to prepare a stock solution of no less than 10 μg/mL of the cytokine. Storage of R&D FGF • Lyophilized samples are stable for up to twelve months from date of receipt at -20° C to -70° C. • Upon reconstitution, this cytokine should be stored under sterile conditions at -20° C to -70° C in a manual defrost freezer for three months without detectable loss of activity. • Avoid repeated freeze-thaw cycles.

R&D Systems Recombinant Human FGF basic (157 aa)FGF basic is a member of the FGF family of at least 23 related mitogenic proteins which show 35 - 60% amino acid conservation. FGF acidic and basic, unlike the other members of the family, lack signal peptides and are apparently secreted by mechanisms other than the classical protein secretion pathway.  This factor contains four cysteine residues, but reduced FGF basic retains full biological activity, indicating that disulfide bonds are not required for this activity. Specifications and Use of R&D FGFR&D FGF Source • A DNA sequence corresponding to the mature human FGF basic protein sequence (Sommer, A. et al., 1987, BBRC 144:543 - 550) was expressed in E. coli. • Note: This is one of multiple forms available for this protein. Check R&D Systems' website, www.RnDSystems.com, for a complete listing of the variants. R&D Recombinant FGF Molecular Mass • The 157 amino acid residue recombinant protein has a molecular mass of 17.4 kDa. Purity of R&D FGF • > 97%, as determined by SDS-PAGE and visualized by silver stain. Endotoxin Level of R&D FGF• < 1.0 EU per 1 μg of the cytokine as determined by the LAL method. R&D FGF basic Activity • Measured by its ability to stimulate proliferation of NR6R-3T3 fibroblasts (Raines, E.W. et al., 1985, Methods Enzym. 109:749). • The ED50 for this effect is typically 0.5 - 2.5 ng/mL in a fluorometric assay using the redox sensitive dye, Resazurin (R&D Systems, Catalog # AR002). Formulation of R&D FGF • Supplied as a 0.2 μm filtered solution in 20 mM Tris-HCl and 1 M NaCl (pH 7.0) containing 50 μg of bovine serum albumin per 1 μg of the cytokine. • Centrifuge the vial before opening to recover entire contents of the vial. Due to possible sublimation during storage, the buffer volume may decrease over time, however, the product is sold by mass and the amount of protein will remain constant. To ensure quantitative recovery, we suggest the stock solution be made in the original vial. R&D FGF Storage • Samples are stable for up to twelve months at -20° C to -70° C in a manual defrost freezer. • Upon thawing, this cytokine can be stored under sterile conditions at 2° - 8° C for one month or at -20° C to -70° C in a manual defrost freezer for three months without detectable loss of activity. • Avoid repeated freeze-thaw cycles.