Human Glial-Derived Neurotrophic Factor Recombinant

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Human Glial-Derived Neurotrophic Factor Recombinant

$70.00$4,700.00


accession P39905


Source Optimized DNA sequence encoding Human GDNF mature chain was expressed in Escherichia Coli.
Molecular weight Native Human Glial-Derived Neurotrophic Factor isgenerated by the proteolytic removal of the signal peptide and propeptide, the molecule has a calculated molecular mass of approximately15 kDa. Recombinant 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 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.1 ng/µg(1EU/µg).
Presentation RecombinantGlial-Derived Neurotrophic Factor was lyophilized from a.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.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.


Interactor Q8N5Y2
Interactor P56159 GFRA1_HUMAN
Molecular function Growth-factor

Methods

Overexpression of EpICD in ROSA GS cells.

GDNF
  • Upregulation of EPCAM by GDNF stimulation.

Surface Plasmon resonance (Biacore) measurements

  • MCer1 was immobilized on a CM5 chip in BIAcore 2000 with an amine group.
  • Binding of Cer1 to hBMP2 , hBMP4, or hGNF (1 µg/ml& ) was analysed in triplicate at 25°C in HBS-P buffer supplemented with 10 mM HEPES, 150 mM NaCl and 0.005% Tween 20 at pH 7.4, with a flow rate of 20 µg/ml min.
  • The kinetics and the dissociation constant (KD) were calculated with BIAevaluation software ver.
  • 4.1 .

Preparation of Primary Hippocampal Cultures

  • Primary hippocampal cultures were prepared from wild-type neonatal (E19) rat embryos (timed pregnant Sprague Dawley rats were obtained from Charles River Laboratories, Wilmington, MA) as described previously

RET51 internalizes more efficiently than RET9.

GDNF
  • Retinoic acid–treated SH-SY5Y cells were serum starved, incubated with or without 100 ng/ml GDNF for 20 min, and lysed.

RET51 internalizes more efficiently than RET9.

100 ng/ml GDNF
  • Retinoic acid–treated SH-SY5Y cells were serum starved, incubated with or without 100 ng/ml GDNF for 20 min, and lysed.

Expression of key growth factors in human endometrium.

GDNF
  • Brown signals for growth factors (EGF, IGF-1, GM-CSF, BDNF, CSF-1, artemin, and GDNF) were found following staining with specific antibodies.

Cell Culture and Biochemical Assays

  • For DmRet phosphorylation assays, cells stably expressing DmRet-3xFLAG or DmRet-3xFLAG and V5-DmGfrlA were plated in 6-well plates at ∼12 M cells/well, in serum-free M3-BPYE medium.
  • Expression was induced with 600 µM CuSO4 for 2 to 4 hours.
  • Recombinant human GDNF was added at 50 ng/ml for 1 hour after which the cells were triturated, washed in PBS, and lysed in membrane lysis buffer supplemented with 1 mM Na2VO4.
  • Insoluble material was sedimented by centrifugation and to the supernatants 0.5 µg of affinity-purified DmRet antibody was added.
  • After 15-minute incubation on ice, protein A-sepharose (GE Healthcare) was added and the samples were rotated in cold for 1–2 hours.
  • The precipitates were washed with the lysis buffer three times, eluted with Laemmli sample buffer, and run on 8% SDS-PAGE.
  • The immunoblots were probed with anti-phosphotyrosine (4G10) and anti-FLAG (M2, Sigma) antibodies.
  • For mammalian RET phosphorylation assay MG87RET cells that…
  • For DmRet phosphorylation assays, cells stably expressing DmRet-3xFLAG or DmRet-3xFLAG and V5-DmGfrlA were plated in 6-well plates at ∼12 M cells/well, in serum-free M3-BPYE medium.
  • Expression was induced with 600 µM CuSO4 for 2 to 4 hours.
  • Recombinant human GDNF was added at 50 ng/ml for 1 hour after which the cells were triturated, washed in PBS, and lysed in membrane lysis buffer supplemented with 1 mM Na2VO4.
  • Insoluble material was sedimented by centrifugation and to the supernatants 0.5 µg of affinity-purified DmRet antibody was added.
  • After 15-minute incubation on ice, protein A-sepharose (GE Healthcare) was added and the samples were rotated in cold for 1–2 hours.
  • The precipitates were washed with the lysis buffer three times, eluted with Laemmli sample buffer, and run on 8% SDS-PAGE.
  • The immunoblots were probed with anti-phosphotyrosine (4G10) and anti-FLAG (M2, Sigma) antibodies.
  • For mammalian RET phosphorylation assay MG87RET cells that stably express human RET51 were transfected with plasmids encoding human GFRα1 or DmGfrlA.
  • RET phosphorylation assay was performed essentially as described previously

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Cell Culture and Biochemical Assays

  • For DmRet phosphorylation assays, cells stably expressing DmRet-3xFLAG or DmRet-3xFLAG and V5-DmGfrlA were plated in 6-well plates at ∼12 M cells/well, in serum-free M3-BPYE medium.
  • Expression was induced with 600 µM CuSO4 for 2 to 4 hours.
  • Recombinant human GDNF was added at 50 ng/ml for 1 hour after which the cells were triturated, washed in PBS, and lysed in membrane lysis buffer supplemented with 1 mM Na2VO4.
  • Insoluble material was sedimented by centrifugation and to the supernatants 0.5 µg of affinity-purified DmRet antibody was added.
  • After 15-minute incubation on ice, protein A-sepharose (GE Healthcare) was added and the samples were rotated in cold for 1–2 hours.
  • The precipitates were washed with the lysis buffer three times, eluted with Laemmli sample buffer, and run on 8% SDS-PAGE.
  • The immunoblots were probed with anti-phosphotyrosine (4G10) and anti-FLAG (M2, Sigma) antibodies.
  • For mammalian RET phosphorylation assay MG87RET cells that…
  • For DmRet phosphorylation assays, cells stably expressing DmRet-3xFLAG or DmRet-3xFLAG and V5-DmGfrlA were plated in 6-well plates at ∼12 M cells/well, in serum-free M3-BPYE medium.
  • Expression was induced with 600 µM CuSO4 for 2 to 4 hours.
  • Recombinant human GDNF was added at 50 ng/ml for 1 hour after which the cells were triturated, washed in PBS, and lysed in membrane lysis buffer supplemented with 1 mM Na2VO4.
  • Insoluble material was sedimented by centrifugation and to the supernatants 0.5 µg of affinity-purified DmRet antibody was added.
  • After 15-minute incubation on ice, protein A-sepharose (GE Healthcare) was added and the samples were rotated in cold for 1–2 hours.
  • The precipitates were washed with the lysis buffer three times, eluted with Laemmli sample buffer, and run on 8% SDS-PAGE.
  • The immunoblots were probed with anti-phosphotyrosine (4G10) and anti-FLAG (M2, Sigma) antibodies.
  • For mammalian RET phosphorylation assay MG87RET cells that stably express human RET51 were transfected with plasmids encoding human GFRα1 or DmGfrlA.
  • RET phosphorylation assay was performed essentially as described previously

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FGSC Culture and Growth Assay

  • To evaluate the growth-promoting activity of each candidate feeder line on the FGSC cultures, 2×105 cells of each feeder line were seeded into individual well of a 12-well plate in triplicate.
  • The candidate feeder cells were irradiated for 8 Krad to generate growth-arrested feeder cells.
  • The FGSCs isolated from ovaries of 18 Tg(ziwi:neo);Tg(ziwi:) transgenic fish (10–12 weeks old) using a published

mRNA expression of the GDNF system in the muscularis propria of the human colon.

GDNF
  • mRNA expression of GDNF and its receptors GFRα1 and RET is significantly down-regulated in patients with DD compared to controls.