Human Platelet Derived Growth Factor-BB Recombinant

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Human Platelet Derived Growth Factor-BB Recombinant

$70.00$3,500.00


accession P01127


Source Optimized DNA sequence encoding Human PDGF-BB mature chain was expressed in Saccharomyces cerevisiae.
Molecular weight Human PDGF-BB, generated by the proteolytic removal of the signal peptide and propeptide, the molecule has a calculated molecular mass of approximately kDa . Recombinant PDGF-BBis a disulfide-linked homodimeric protein consisting of two109 amino acid residue subunits. Recombinant PDGF-BB migrates due to glycosylation as an approximately32 kDa protein under non-reducing conditions andaskDa under 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 proliferation of Balb/cT3 cells.The expected ED50 for this effect is.0-3.0 ng/ml, corresponding tox106 IU/mg.
Protein Sequence MNRCWALFLS LCCYLRLVSA EGDPIPEELY EMLSDHSIRS FDDLQRLLHG DPGEEDGAEL DLNMTRSHSG GELESLARGR RSLGSLTIAE PAMIAECKTR TEVFEISRRL IDRTNANFLV WPPCVEVQRC SGCCNNRNVQ CRPTQVQLRP VQVRKIEIVR KKPIFKKATV TLEDHLACKC ETVAAARPVT RSPGGSQEQR AKTPQTRVTI RTVRVRRPPK GKHRKFKHTH DKTALKETLG A
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 PlateletDreived GrowthFactor-BB was lyophilized from a.2 μm filtered.02M acetic acid solution 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 P01730 CD4_HUMAN
Interactor P16234 PGFRA_HUMAN
Interactor P09619 PGFRB_HUMAN
Molecular function Developmental-protein
Molecular function Growth-factor
Molecular function Mitogen

Methods

  • MSCs were placed into step one medium, which consists of DMEM supplemented with SPN, 2mM L-glutamine, 20ng/ml human epidermal growth factor (hEGF, R&D Systems, Minneapolis, MN, ), 20ng/ml human basic fibroblast growth factor (hbFGF, R&D Systems) and 10μL/ml N2 supplement (insulin 5μg/ml, progesterone 20nM, putrescin 100μM, selenium 30nM, transferrin 100μg/ml, , , ).
  • 72 hours later, the MSCs were placed into step two medium consisting of DMEM supplemented with SPN, 2mM L-glutamine, 1mM dibutyryl cyclic AMP (dbcAMP), 0.5mM isobutylmethylxanthine (, from), 20 ng/ml hbFGF, 50ng/ml human neuregulin1-β1 and 5 ng/ml Platelet-Derived Growth Factor-AA (PDGF).
  • MSCs grown in medium'>serum-free medium containing DMEM, glutamine and SPN served as untreated controls.
  • Due to species-specific toxicity, when inducing mouse MSCs the concentrations of IBMX and cAMP were halved in the step two medium.

Cell Culture

  • H1 (WA01) cells from WiCell were expanded in ES cell growth media and differentiated according to our laboratory’s established protocols EBs) were formed by suspending undifferentiated ES cell colonies in ES growth media on ultra low adhesion culture dishes.
  • The neural differentiation was commenced by growing EBs in medium'>N2B27 medium NPs).
  • NPs were then passaged with 0.05% trypsin/ETA and cultured for 3 weeks in N2B27 media with 20 ng/ml of PGF-AA and 20 ng/ml EGF until transplantation.
  • A human fibroblast line (HFF1, from ATCC) from neonate foreskins were cultured in 10% fetal bovine serum as previously described

Cell culture

  • Immortalized p19ARF-deficient MIM-1-4 hepatocytes were grown on collagen-coated culture dishes in RPMI 1640 plus 10% fetal calf serum, 40 ng/ml human TGF-α , 30 ng/ml human insulin-like growth factor II , 1.4 nM insulin and antibiotics, as described previously (2 and routinely screened for the absence of mycoplasma.

Primary oligodendrocyte-microglia-co-culture

  • Primary OL and microglia cells (MG) were prepared from the cerebral hemispheres of Sprague Dawley rats at P1 to P2 using a shaking method of mixed-glia-cultures 2 for 3 days in a medium'>serum-free medium'>basal-defined medium (BDM: DMEM , 0.1% bovine serum albumin , 50 µg/ml human apo-transferrin , 50 µg/ml insulin , 30 nM sodium selenite , 10 nM D-biotin , 10 nM hydrocortisone , 10 ng/ml human recombinant platelet-derived growth factor , and 10 ng/ml human recombinant basic fibroblast growth factor before MG were added.
  • Cultures were kept in a humidified incubator at 37°C and 5% CO2.
  • Purified OL contained <5% astrocytes and <1% microglia and purified microglia contained <0.1% astrocytes and <1% oligodendrocytes.
  • OL-MG-ratio was set to 1∶1 following cell count results in the rat brain

Differentiation to a Schwann cell phenotype

  • Human ADSCs were induced into neurospheres.
  • Briefly, we harvested human ADSCs (80–90% confluence) and then plated them in plastic dish a concentration of 1–2 × 105/cm2 in DMEM: F12 supplemented with 20 ng/ mL EGF , 20 ng/mL basic fibroblast growth factor (bFGF) and 2% B27 (1:50) at 37°C in 5%CO2.
  • We added fresh medium every 3 to 4 days.
  • After 7 days, neurospheres were triturated using a fire-polished Pasteur pipette and re-plated in Laminin coated six-well chamber slides contain DMEM: F12 supplemented with 10% FBS, 14 μM forskolin , 5 ng/mL platelet-derived growth factor-AA (PDGF, ), 10 ng/mL bFGF and 200 ng/mL recombinant human heregulin-beta1 (HRG) for terminal differentiation.
  • The cells were incubated for 9 days under these conditions, and then harvested for investigation.

Chemotactic response of St-T1b cells or primary hESCs to PDGF-BB, HB-EGF and trophoblast conditioned medium.

PDGF-BB
  • The bottom reservoir contained MM1-10% (controls), PDGF-BB, HB-EGF or conditioned medium from the trophoblast cell line AC-1M88 undiluted (100%) or diluted to 80% or 20%.

P-Rex1 requires a factor present in serum in order to drive alterations in cell shape and invasion; PDGF can substitute for serum to drive invasion of P-Rex1 expressing cells.

recombinant human PDGF-BB
  • The invasive migration of vector control (pLHCX) or P-Rex1 expressing fibroblasts into Matrigel in the absence or presence of a serum (0–10%) or PDGF-BB (0–50 ng ml−1) gradient was assayed and quantified as for Figure 3B (±SEM, ***P<0.0005, Mann-Whitney rank sum test, 3 independent experiments).

Influence of selected cytokines and growth factors on the IL-33 synthesis in RA-SFs.

PDGF-BB
  • RA-SFs (n=4) were stimulated for 24 h with TNF-α, IL-1β, IL-18, PDGF-BB or TGF-1β (concentrations as indicated in the figure).

Influence of selected cytokines and growth factors on the IL-33 synthesis in RA-SFs.

PDGF-BB
  • RA-SFs (n=4) were stimulated for 24 h with TNF-α, IL-1β, IL-18, PDGF-BB or TGF-1β (concentrations as indicated in the figure).

Isolation and culture of glioma cells

  • Glioma biopsies of fresh tumor tissue were obtained from patients operated at the Clinic of Neurosurgery, Lund University Hospital, Sweden.
  • Written informed consent was obtained from patients.
  • For preparing viable glioma cells, the fresh specimen were cut into small pieces, and incubated in IMDM with 0.5 mg/ml collagenase and 25 mg/ml DNAse at 37°C for 40 minutes.
  • Red cells were lysed with NH4Cl.
  • The remaining glioma cells were washed in PBS containing 2% fetal calf serum (FCS).
  • Glioma cells were grown in poly-L-Lysine (PLL) coated flasks with medium'>MEM/F12 (1∶1) medium supplemented with 2% FCS, --Glucose solution (0.6%), heparin (5 µg/ml), sodium bicarbonate (0.1%), N2 supplement , FGF2 (20 ng/ml), sonic hedgehog (SHH) (2 ng/ml of C24II version& ), and PGF-AA(20 ng/ml).