Human Granulocyte-Colony Stimulating Factor Recombinant

HomeHematopoietins, IL-6 gp130 family, Recombinant Human Cytokines

Human Granulocyte-Colony Stimulating Factor Recombinant

$70.00 – $4,700.00

SKU: RKP09919 Categories: Hematopoietins, IL-6 gp130 family, Recombinant Human Cytokines Tags: C17orf33, CSF3, Filgrastim, g-csf, gcsf, Lenograstim, P09919, Pluripoietin

Description

Methods (2)


accession	P09919

Source	DNA sequence encoding the extracellular domain of Human CSF3 was expressed in E. coli.
Molecular weight	Recombinant Human CSF3 is a protein consisting of amino acid residues [Ala 30-Pro204] ,and migrates as an approximately 19 kDa protein on SDS-PAGE.
Purity	>95%, as determined by SDS-PAGE and HPLC
Biological Activity	The ED(50) was determined by the dose-dependent proliferation of murine M-NFS-60 cells is < 0.1 ng/ml, corresponding to a specific activity of ≥ 1x 10 ⁸ units/mg.
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	Recombinant CSF3 was lyophilized from 0.2 µm filtered PBS solution pH7.4
Sequence	ATPLGPASSL PQSFLLKCLE QVRKIQGDGA ALQEKLVSEC ATYKLCHPEE LVLLGHSLGI PWAPLSSCPS QALQLAGCLS QLHSGLFLYQ GLLQALEGIS PELGPTLDTL QLDVADFATT IWQQMEELGM APALQPTQGA MPAFASAFQR RAGGVLVASH LQSFLEVSYR
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.
Usage	This product is for research purposes only.It may not be used for therapeutics or diagnostic purposes.

Interactor	P40223
Molecular function	Cytokine
Molecular function	Growth-factor

Methods


Bioactivity Assay The proliferation assay of NFS-60 cells in response to the presence of hG-CSF measured by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] method was used to test the bioactivity of expressed hG-CSF in plants. NFS-60 cells were cultured with medium'>RPMI-1640 medium supplemented with 10% fatal bovine serum (FBS) and commercial recombinant hG-CSF (2 ng/ml) and kept under 5% CO₂at 37 °C in a humidified condition. For MTT test, total soluble protein extracted from leaves and seeds (see Protein extraction and immunoblot above) were used for analysis and NFS-60 cells (1 × 10⁴per well) were cultured in a 96 well plate and treated with the medium medium'>(RPMI-1640 medium supplemented with 10% FBS) containing the following samples: commercial recombinant hG-CSF (1 ng); total soluble protein extracted from leaf (TSP-L) and seed (TSP-S) of transgenic plant harboring the Construct USH containing 1… The proliferation assay of NFS-60 cells in response to the presence of hG-CSF measured by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] method was used to test the bioactivity of expressed hG-CSF in plants. NFS-60 cells were cultured with medium'>RPMI-1640 medium supplemented with 10% fatal bovine serum (FBS) and commercial recombinant hG-CSF (2 ng/ml) and kept under 5% CO₂at 37 °C in a humidified condition. For MTT test, total soluble protein extracted from leaves and seeds (see Protein extraction and immunoblot above) were used for analysis and NFS-60 cells (1 × 10⁴per well) were cultured in a 96 well plate and treated with the medium medium'>(RPMI-1640 medium supplemented with 10% FBS) containing the following samples: commercial recombinant hG-CSF (1 ng); total soluble protein extracted from leaf (TSP-L) and seed (TSP-S) of transgenic plant harboring the Construct USH containing 1 ng expressed hG-CSF; TSP-L and TSP-S from SH plant, containing the same amount of total protein as the USH samples while 0.8 and 0.9 ng expressed hG-CSF, respectively; TSP-L and TSP-S from transgenic plant harboring empty pBI121 plasmid (denoted as EP), containing the same amount of total protein as the USH samples; TSP-L and TSP-S from EP plant (as above) supplemented with 1 ng commercial hG-CSF; TSP-L and TSP-S from wildtype (WT) plants, containing the same amount of total protein as the USH samples; TSP-L and TSP-S fromWT plant (as above) supplemented with 1 ng commercial hG-CSF; extraction buffer (, in supplement with protein inhibitor); extraction buffer supplemented with 1 ng commercial hG-CSF. All treatments, including the untreated control, were carried out in triplicate. Cells were incubated at 37 °C for 72 hours. Finally, 20 μl of MTT (5 mg/ml) was added and the samples were left to incubate at 37 °C for 4 h. The medium was discarded and the formazan dye was dissolved in DMSO (100 μl) at 37 °C for 30 min. Absorbance was measured for all samples at 550 nm using a VICTOR3 V™ Multilabel Counter . Read more
CD34 Cord blood (CB) samples were obtained from the Hannover Medical School following written consent of the donors as approved by the Hannover Medical School local ethics committee. Total nucleated cells were isolated by a Ficoll gradient followed by enrichment for CD34⁺ cells employing MACS purification . Isolated cells were frozen until further usage. CB-CD34⁺ cells were cultured in medium'>StemSpan medium supplemented with 1% penicillin/streptomycin and one of four cytokine combinations using hSCF, hTHPO, hFLT3-L, hGCSF, hIGFBP2, hAngptl5, hFGF-1, and hIL6 . The StemRegenin compound was used at a concentration of 1 µmol/l as described.