Human Epidermal Growth Factor Recombinant

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Human Epidermal Growth Factor Recombinant

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accession P01133


Source Optimized DNA sequence encoding HumanEGF mature chain was expressed in Escherichia Coli.
Molecular weight Native human EGF, generated by the proteolytic removal of the signal peptide and propeptide,the molecule has a calculated molecular mass of approximately6 kDa. RecombinantEpidermal Growth Factoris a disulfide-linked monomer protein consisting of54 amino acid residue subunits, migrates as an approximately6 kDa protein under non-reducing and reducing conditions in SDS-PAGE.
Purity >98%, as determined by SDS-PAGE and HPLC
Biological Activity The ED50 was determined by a cell proliferation assay using balb/c 3T3 cells is ≤ 0.5 ng/ml, corresponding to a specific activity of ≥2 x 107 units/mg.
Protein Sequence 970 980 990 1000 1010 1020 REDDHHYSVR NSDSECPLSH DGYCLHDGVC MYIEALDKYA CNCVVGYIGE RCQYRDLKWW 1030 1040 1050 1060 1070 1080 ELR HAGHGQQ QKVIVVAVCV VVLVMLLLLS LWGAHYYRTQ KLLSKNPKNP YEESSRDVRS 1090 1100 1110 1120 1130 1140 RRPADTEDGM SSCPQPWFVV IKEHQDLKNG GQPVAGEDGQ AADGSMQPTS WRQEPQLCGM 1150 1160 1170 1180 1190 1200 GTEQGCWIPV SSDKGSCPQV MERSFHMPSY GTQTLEGGVE KPHSLLSANP LWQQRALDPP HQMELTQ
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 RecombinantEpidermal Growth Factor was lyophilized from a 0.2μm filtered concentrated (1mg/ml) solution in PBS, pH 7.2.
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 2 years from date of receipt at -20° C. Upon reconstitution, this cytokine can be stored in working aliquots at 2° - 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 P00533 EGFR_HUMAN
Molecular function Growth-factor

Methods

Identification of an ERK-aconitase protein complex that is disrupted by aconitase inhibition.

EGF
  • mAcon-FLAG was coexpressed with ERK2 WT or D319N in HEK293T cells ±500 µM FA, ±100 ng/ml EGF, followed by IP and IB as in panel A.

Neural progenitor cell isolation and maintenance

  • All procedures were performed in sterile fashion in a class II biosafety cabinet.
  • A representative portion (2.5 cm) of three regions (thoracic, cervical and lumbar) of the spinal cord was used for Neural Progenitor Cell (NPC) isolation as described previously.
  • Briefly, tissue was diced and a single cell suspension was obtained by enzymatic dissociation of the tissue at 37 °C for approximately 30–40 minutes with 2.5 U/ml papain , 250 U/ml of DNase I , and 1 U/ml neutral protease .
  • After dissociation, the cell suspension was mixed with DMEM/F12 with 10% fetal bovine serum (FBS, , ), passed through a 70 μM filter, and centrifuged.
  • The cell pellet was resuspended in DMEM/F12 with 10% FBS and combined 1:1 with percoll (GE Healthcare, Piscataway, NJ).
  • The cell/percoll mixture was centrifuged at 20,000 g for 30 min at room temperature and the low buoyancy fraction (10 ml) above the red blood cell layer…
  • All procedures were performed in sterile fashion in a class II biosafety cabinet.
  • A representative portion (2.5 cm) of three regions (thoracic, cervical and lumbar) of the spinal cord was used for Neural Progenitor Cell (NPC) isolation as described previously.
  • Briefly, tissue was diced and a single cell suspension was obtained by enzymatic dissociation of the tissue at 37 °C for approximately 30–40 minutes with 2.5 U/ml papain , 250 U/ml of DNase I , and 1 U/ml neutral protease .
  • After dissociation, the cell suspension was mixed with DMEM/F12 with 10% fetal bovine serum (FBS, , ), passed through a 70 μM filter, and centrifuged.
  • The cell pellet was resuspended in DMEM/F12 with 10% FBS and combined 1:1 with percoll (GE Healthcare, Piscataway, NJ).
  • The cell/percoll mixture was centrifuged at 20,000 g for 30 min at room temperature and the low buoyancy fraction (10 ml) above the red blood cell layer was collected.
  • Cells were washed and resuspended in medium'>NPC medium containing DMEM/F12 supplemented with 10% FBS , 10% BIT9500 ( Cell , , ), 1% N2 supplement , 20 ng/ml of FGF-2 , 20 ng/ml of EGF , and 20 ng/ml of PDGF-AB .
  • NPCs were cultured on fibronectin coated plates and after 24 hours, the media was replaced with serum-free NPC medium.
  • Half of the medium was subsequently replaced every 2 days.
  • Cells were passed when 60–70% confluence was reached in about 3–4 weeks.

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Isolation of epidermal stem cells and cell expansion

  • Human foreskin samples were derived from voluntary circumcisions with informed consents and the protocol was approved by the Ethical Committee of the Institute of Zoology, Chinese Academy of.
  • Human epidermal stem cells (hEpSCs) were isolated and expanded using our previous described method

Cell lines

  • 081110 and 081024 non-adherent neurosphere lines were produced in our laboratory from human tumor samples received immediately following resection from consented GBM patients.
  • Neurospheres were cultured in NeuroCult NS-A basal medium supplemented with 10ng/mL FGF , 10ng/mL EGF , and 0.0004% heparin .
  • HCT116 cells and HCT116 lines containing only the wild-type (HPIK3CA WT #123) or mutant (HPIK3CA MT #125) PIK3CA alleles were obtained from Dr. Bert Vogelstein (Johns Hopkins), and were grown in McCoy's Modified 5A medium (Sigma-Aldrich) supplemented with 10% FBS.
  • D54 and SKMG26 cells were grown in DMEM supplemented with 10% FBS.

Cell culture

  • GM97, GM1600, and GM1605 were established from surgically resected specimens from glioblastoma patients at the University of California Los Angeles, in accordance with protocols approved by the University of California Los Angeles Institutional Review Board, as previously described 2 cell culture incubator .
  • Glioblastoma and medulloblastoma cells grown as sphere cultures were collected by gentle centrifugation (800×g, 5 min) and trypsinized with 0.05% TrypLESelect for 5 min by pipetting.
  • Cells were washed twice with phosphate buffered saline (PBS), counted, and seeded at a density of 2000 cells per 200 µL into 96-well ultralow-attachment plates (Corning).