Human Glial-Derived Neurotrophic Factor Recombinant
Categories: Neurotrophic factorsRecombinant Human Cytokines$70.00 – $4,700.00
Description
Accession
P39905
Source
Optimized DNA sequence encoding Human GDNF mature chain was expressed in Escherichia Coli.
Molecular weight
Native Human Glial-Derived Neurotrophic Factor is generated by the proteolytic removal of the signal peptide and propeptide, the molecule has a calculated molecular mass of approximately 15 kDa. Recombinant GDNF is a disulfide-linked homodimeric protein consisting of two 125 amino acid residue subunits,and migrates as an approximately 30 kDa protein under non-reducing conditions and as 15 kDa under reducing conditions in SDS-PAGE.
Purity
>95%, as determined by SDS-PAGE and HPLC
Biological Activity
The ED(50) was determined by theproliferation of rat C6 cells is <.1 ng/ml, corresponding to a specific activity of > x units/mg.
Protein Sequence
MKLWDVVAVC LVLLHTASAF PLPAGKRPPE APAEDRSLGR RRAPFALSSD SNMPEDYPDQ FDDVMDFIQA TIKRLKRSPD KQMAVLPRRE RNRQAAAANP ENSRGKGRRG QRGKNRGCVL TAIHLNVTDL GLGYETKEEL IFRYCSGSCD AAETTYDKIL KNLSRNRRLV SDKVGQACCR PIAFDDDLSF LDDNLVYHIL RKHSAKRCGC I
Endotoxin
Endotoxin content was assayed using a LAL gel clot method. Endotoxin level was found to be less than 0.1 ng/µg(1EU/µg).
Presentation
RecombinantGlial-Derived Neurotrophic Factor was lyophilized from a 0.2 μm filtered solution in.5% glycine,.5% sucrose,.01% Tween80, mM Glutamic acid, pH.5.
Reconstitution
A quick spin of the vial followed by reconstitution in distilled water to a concentration not less than 0.1 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 at2° -8° 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.
Interactor
Q8N5Y2
Interactor
Molecular function
Methods
Human motor neuron differentiation
- Human embryonic stem cells (hESCs, lines H9, passages 19 to 35) were used to generate neuroectodermal cells.
- Motor neuron induction was carried out as previously described .
- Briefly, hESC–derived neuroectodermal cells were treated with retinoic acid (0.1 μM) for caudalization for one week in a chemically defined neural medium (NM: DMEM/F12, nonessential amino acids, 2 μg/ml heparin, and the neural cell supplement N2 .
- The neuroepithelial clusters were isolated and suspended in the NM in the presence of both retinoic acid and purmorphamine (1 μM).
- Purmorphamine was removed from the NM one week later.
- The formed progenitor spheres were subsequently cultured on glass coverslips coated with polyornithine and laminin (2–4 clusters/coverslip in a 24-well plate) in the presence of 0.5 ml NM, supplemented with BNF (10 ng/ml), GNF(10 ng/ml& ), IGF1 (10 ng/ml), cAMP (1 μM), ascorbic acid (200 ng/ml, Cell ), and 50 nM…
SSC cultures
- SSC medium was composed of StemPro-34 SFM with the following supplements: StemPro supplement , 1× N2 supplement , 6 mg/ml d--glucose , 30 mg/ml pyruvic acid , 1 μl/ml DL-lactic acid , 5 mg/ml bovine serum albumin (BSA), 1% fetal bovine serum , 2 mM L-glutamine , 50 μM β-mercaptoethanol , 1 × penicillin/streptomycin , 1× minimal essential medium (MEM) non-essential amino acids , 1× MEM vitamins , 30 ng/ml β-estradiol , 60 ng/ml progesterone , 20 ng/ml human EGF , 20 ng/ml human bFGF , 20 ng/ml human GDNF , and 103 U/ml murine leukemia inhibitory factor .
SSC cultures
- SSC medium was composed of StemPro-34 SFM with the following supplements: StemPro supplement , 1× N2 supplement , 6 mg/ml d--glucose , 30 mg/ml pyruvic acid , 1 μl/ml DL-lactic acid , 5 mg/ml bovine serum albumin (BSA), 1% fetal bovine serum , 2 mM L-glutamine , 50 μM β-mercaptoethanol , 1 × penicillin/streptomycin , 1× minimal essential medium (MEM) non-essential amino acids , 1× MEM vitamins , 30 ng/ml β-estradiol , 60 ng/ml progesterone , 20 ng/ml human EGF , 20 ng/ml human bFGF , 20 ng/ml human GDNF , and 103 U/ml murine leukemia inhibitory factor .
Stability of free versus conjugated neurotrophic factors at 37°C in the absence ((a1), (b1), and (c1)) and in the presence ((a2), (b2), and (c2)) of cells.
- In the upper row free or conjugated neurotrophic factors (GDNF, βNGF, and FGF-2) were added to culture medium, each type separately (10 ng/mL, final concentration), and placed at 37°C (in the absence of cells).
Schematic illustration of culture models used in this study.
- Abbreviations: np, nanoparticle; NTFs, neurotrophic factors; DRG, dorsal root ganglion; SFM, serum-free medium; NGF, nerve growth factor; FGF, fibroblast growth factor; GDNF, glia-derived neurotrophic factor; BMSCs, bone marrow-derived mesenchymal stromal cells; EGFP, enhanced green fluorescent protein; PC-12 cells, cell line from rat pheochromocytoma cells; PSN, penicillin–streptomycin–neomycin; BSA, bovine serum albumin; DMEM, Dulbecco’s Modified Eagle’s Medium; MEM, Minimum Essential Medium; FCS, fetal calf serum; pen/strep, penicillin/streptomycin.
Schematic illustration of culture models used in this study.
- Abbreviations: np, nanoparticle; NTFs, neurotrophic factors; DRG, dorsal root ganglion; SFM, serum-free medium; NGF, nerve growth factor; FGF, fibroblast growth factor; GDNF, glia-derived neurotrophic factor; BMSCs, bone marrow-derived mesenchymal stromal cells; EGFP, enhanced green fluorescent protein; PC-12 cells, cell line from rat pheochromocytoma cells; PSN, penicillin–streptomycin–neomycin; BSA, bovine serum albumin; DMEM, Dulbecco’s Modified Eagle’s Medium; MEM, Minimum Essential Medium; FCS, fetal calf serum; pen/strep, penicillin/streptomycin.
