Mouse Glial-Derived Neurotrophic Factor Recombinant

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Mouse Glial-Derived Neurotrophic Factor Recombinant

$70.00$4,700.00


accession P48540


Source Optimized DNA sequence encodingMouse Glial Derived Neurotrophic Factor mature chain was expressed in Escherichia Coli.
Molecular weight NativeMouse GDNF isgenerated by the proteolytic removal of the signal peptide and propeptide, the molecule has a calculated molecular mass of approximately15 kDa. Recombinant mouse GDNF is a disulfide-linked homodimeric protein consisting of two amino acid residue subunits,and migrates as an approximately30 kDa protein under non-reducingconditions and askDa 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 PLPAGKRLLE APAEDHSLGH RRVPFALTSD SNMPEDYPDQ FDDVMDFIQA TIKRLKRSPD KQAAALPRRE RNRQAAAASP ENSRGKGRRG QRGKNRGCVL TAIHLNVTDL GLGYETKEEL IFRYCSGSCE SAETMYDKIL KNLSRSRRLT SDKVGQACCR PVAFDDDLSF LDDNLVYHIL RKHSAKRCGC I
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 GDNF was lyophilized from a.2 μm filtered PBS solution.
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.


Molecular function Growth-factor

Methods

Neural precursor culture and viral transduction

  • To differentiate NPs to neurons, NPs were plated on polyornithine and laminin coated plates in NP media and grown until they reached 70% confluence.
  • FGF was removed and NP media supplemented with 20 ng/ml BDNF , 20 ng/ml GDNF and 0.5 mM dibutyryl cAMP (N6,2′-O-Dibutyryladenosine 3′,5′-cyclic monophosphate sodium salt).
  • Medium was exchanged every 2–3 days.

Monolayer neuronal differentiation

  • iPSC cultures were dissociated with Accutase to generate single-cell suspensions, then differentially plated on gelatin-coated plasticware for 1 h in medium'>hESC medium in the presence of ROCK inhibitor (Y27632, Ascent) to remove SNL feeders.
  • The nonattached cells were resuspended in MEF-CM (&systems) with 10 ng ml−1 FGF2 and plated on growth-factor reduced Matrigel (B) at a density of 25,000 cells cm−2 and allowed to propagate in self-renewal conditions for 72 h or until 70–90% confluent, whereupon media was changed to KS medium containing 50 ng ml−1 Noggin , 10 μM −1 SHH C24II and 50 ng ml−1 Wnt1 for the second day onwards.
  • kk1 blocking antibody (100 ng ml−1& ) was also added for the second day only.
  • After 5 days, medium'>medium'>KSR medium'>medium containing these ligands was cross-tapered with medium'>N2B27 medium'>medium (Cells) containing…
  • iPSC cultures were dissociated with Accutase to generate single-cell suspensions, then differentially plated on gelatin-coated plasticware for 1 h in medium'>hESC medium in the presence of ROCK inhibitor (Y27632, Ascent) to remove SNL feeders.
  • The nonattached cells were resuspended in MEF-CM (&systems) with 10 ng ml−1 FGF2 and plated on growth-factor reduced Matrigel (B) at a density of 25,000 cells cm−2 and allowed to propagate in self-renewal conditions for 72 h or until 70–90% confluent, whereupon media was changed to KS medium containing 50 ng ml−1 Noggin , 10 μM −1 SHH C24II and 50 ng ml−1 Wnt1 for the second day onwards.
  • kk1 blocking antibody (100 ng ml−1& ) was also added for the second day only.
  • After 5 days, medium'>medium'>KSR medium'>medium containing these ligands was cross-tapered with medium'>N2B27 medium'>medium (Cells) containing the same ligands over 7 days (75% medium'>medium'>KSR and 25% medium'>N2B27 first day, 50% of each third day, and 25% medium'>medium'>KSR with 75% medium'>N2B27 fifth day).
  • Once established in N2B27 conditions, Wnt1, Noggin, SB431542 and −1 BDNF , 0.2 mM ascorbic acid and 100 ng ml−1 FGF8 were added.
  • Three days later, cells were dissociated with Hank's buffered saline solution for 1 h at room temperature, and lifted mechanically, then replated en bloc on to poly-l-ornithine/laminin-coated plasticware.
  • Neuronal maturation ensued with BDNF and ascorbic acid as before, supplemented with 10 ng ml−1 GDNF , 1 ng ml−1 TGFβ3 and 0.5 mM dibutyryl-cAMP for the next 7 days.
  • After a total of 23–31 days, the resulting neuronal cultures were analysed by immunocytochemistry, qPCR and western blot.

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The SCG SC development assay is free of GDNF.

recombinant murine GDNF
  • Western Blot for GDNF with different dilutions of rat-tail collagen (20μl, 10μl, 5μl, 2.5μl, 1.25μl and 0.6125μl collagen) were analyzed using anti-GDNF antibody specific for human and rat GDNF.

Alterations of gene expression of neurotrophins in the DRG after HSV-1 infection.

GDNF
  • Gene expression levels of ATF3 and neurotrophins (NGF, GDNF, BDNF, and NT3) were normalized to the GAPDH level, and are presented as the fold change in the ratio of the affected side to the contralateral one.

Differentiation of smNPCs

  • For generation of more ventral CNS neurons, including mDANs, medium'>smNPC expansion medium was changed 2 days after splitting to N2B27 medium with 100 ng/mL FGF8 , 1 µM PMA, and 200 µM AA.
  • After 8 days in this medium, maturation medium–N2B27 with 10 ng/mL BDNF , 10 ng/mL GDNF , 1 ng/mL TGF-b3 , 200 µM AA, and 500 µM dbcAMP–was used for the maturation of neurons.
  • 0.5 µM PMA was added to this medium for 2 more days.
  • One day after changing to maturation medium, the cultures were split at a 1∶3 ratio as small clumps, or single cells after Accutase treatment, or earlier when cultures became over-confluent.
  • Cultures were analyzed after 2 weeks in maturation conditions unless otherwise indicated.