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Human Keratinocyte Growth Factor Recombinant

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$70.00$4,700.00

SKU: RKP21781 Tags: , , , ,

Description

Accession
P21781
Source
Optimized DNA sequence encoding HumanKeratinocyte Growth Factor mature chain was expressed in Escherichia Coli.
Molecular weight
Native human KGF is generated by the proteolytic removal of the signal peptide and propeptide, the molecule has a calculated molecular mass of approximately kDa. Recombinant Keratinocyte Growth Factoris a monomeric protein consisting of amino acid residue subunits. Interefron alphab migrates as an approximately kDa protein under non-reducing conditions and reducing conditions in SDS-PAGE.
Purity
>96%, as determined by SDS-PAGE and HPLC
Biological Activity
The ED(50) was determined by the dose-dependent proliferation ofBAF3 cells was found to be less than10 ng/ml.

Protein Sequence
MHKWILTWIL PTLLYRSCFH IICLVGTISL ACNDMTPEQM ATNVNCSSPE RHTRSYDYME GGDIRVRRLF CRTQWYLRID KRGKVKGTQE MKNNYNIMEI RTVAVGIVAI KGVESEFYLA MNKEGKLYAK KECNEDCNFK ELILENHYNT YASAKWTHNG GEMFVALNQK GIPVRGKKTK KEQKTAHFLP MAIT
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 KGF was lyophilized from a.2 μm filteredM NaCl,mM PBsolution pH7.0.
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
Interactor
Molecular function
Molecular function
Molecular function

Methods

HaCaT Cells

  • HaCaT cells, an immortalized human keratinocyte line (kind gift of Dr P. Boukamp; 2.
  • Cells were passaged weekly in T75 flasks and all the experiments were performed with sub-confluent cells.
  • HaCaT cells at 70% confluence were stimulated with hFGF7, hFGF10 or hFGF22 at a concentration of 100 ng/ml, co-treated with(300 ng/ml).
  • FGF dependent stimulation was blocked by 30 minute pre-treatment with the FGFR specific inhibitor, PD173074 (1.7 µM).

Human cell cultures

  • Primary human type II alveolar epithelial (ATII) cells were seeded on Matrigel®-coated transwell filters and maintained using Bronchial Epithelial Medium enhanced with the Bullet kit supplements provided by the manufacturer (BEGM, , ) supplemented with 10% charcoal-stripped FBS and 10 ng/mL of keratinocyte growth factor (KGF, , ).
  • ATII cells were grown to confluence (4–5 days) then used for experiments.
  • Before each experiment, the A549 or ATII cells were washed 2X with appropriate medium containing no serum or antibiotics.
  • The medium'>serum-free medium used during experimentation ensured no interference by serum proteins and did not adversely affect the health or metabolic activity of the cells.

Induced pluripotent stem cell-directed differentiation

  • For hepatocyte differentiation, post-excised iPSCs were grown on Matrigel as stated above until reaching a 60 to 70% confluence upon which endoderm induction was initiated by replacing the post-excised iPSCs for 24 hours with RPMI 1640 medium , supplemented with 0.5 mg/ml albumin fraction V , and 100 ng/ml Activin A .
  • On the following 2 days, 0.1 and 1% insulin–transferrin–selenium were added to the medium, respectively.
  • Post-excised iPSCs were then cultured in hepatocyte culture medium containing 30 ng/ml fibroblast growth factor-4 and 20 ng/ml BMP2 for 4 days.
  • The now-differentiated cells were then incubated in hepatocyte culture medium'>medium containing 20 ng/ml hematopoietic growth factor and 20 ng/ml keratinocyte growth factor for 6 days, in hepatocyte culture medium'>medium containing 10 ng/ml oncostatin-M plus 0.1 μM dexamethasone…

hESC and iPSC Culture and Differentiation

  • For thymic endoderm differentiation assays, cells were initially cultured in APEL or BPEL medium (−1 human Activin A.
  • On day 5A-containing APEL or BPEL medium was replaced with APEL or BPEL medium alone.
  • On day 7, EBs were transferred to 96-well flat-bottom adherent plates in AEL or BEL medium (media lacking PVA) (−1 human keratinocyte growth factor (KGF& ) was used to replenish the cultures on days 14, 21, 28, and 35.
  • Embryoid bodies were harvested for analysis by flow cytometry at the times indicated.
  • For experiments using MIXL1 hESCs (−1 Activin A, 20 ng ml−1 human BMP4, and 100 ng ml−1 Activin A, or with 100 ng ml−1 FGF2 .
  • In all instances, hESC and iPSC cultures and differentiations were maintained at 37°C, in a 5% CO2/air environment.
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