Recombinant human FGF-10 protein (Qk003)
Fibroblast growth factor 10 (FGF-10) is a member of the FGF family of heparin binding glycoproteins involved in a number of different embryo and adult cell and tissue types, including mesenchymal, neuronal and epithelial cells. FGF-10 is expressed in the mesenchyme and functions through interacting with the epithelial FGF Receptor 2b (Fgfr2b) with heparin/heparan sulfate 1. It has also been shown to interact weakly with FGF Receptor 1b2. The mature form of human FGF-10 is an approximately 20 kDa protein highly similar to FGF-7 and with a serine-rich region near its N-terminus3. It is secreted by mesenchymal cells and is bound and activated by extracellular FGF-BP4.
FGF-10 is first active in the limb bud mesoderm where it creates and maintains FGF signalling with epithelial FGF-8, then drives a positive feedback loop accumulating mesenchyme in the growing bud, and finally induces the apical ectodermal ridge which ultimately gives rise to feet and hands5. Lung development is based on the same epithelial-mesenchymal FGF mediations involving FGF-10 from the foregut mesenchyme signalling to FGFR-2 in the foregut epithelium6. Further, FGF-10 is involved in the development of white adipose tissue, heart, liver, brain, kidney, thymus, inner ear, tongue, trachea, eye, prostate, salivary gland, and mammary gland, and has been shown to induce migration and invasion of pancreatic cancer cells, to be associated with breast cancer risk, and patients with FGF-10 haploinsufficiency present symptoms of chronic obstructive pulmonary disease. FGF-10 also drives the differentiation of embryonic stem cells into gut-like structures, cardiomyocytes, and hepatocytes1.
Summary: Mature domain of human FGF10 (residues 64-208, Uniprot: O15520) expressed in E.coli and purified to homogeneity. Mature protein is a non-glycosylated protein with a molecular mass of 17 kDa
Molecular mass: ~17 kDa
Form: protein is provided frozen in PBS (carrier protein-free). Protein concentration is 1 mg/ml.
Thaw the sample on ice, spin briefly and dilute with PBS as needed. Our protein are supplied carrier-protein free. If compatible with your work, add carrier protein of your choice such as BSA, HSA or gelatin to further minimize loss by adsorption. Spin in a microfuge for 5 minutes at maximum speed, and divide the solution into suitable aliquots and store at -80°C. We recommend that single-use aliquots should be prepared to avoid freeze-thaw cycles.
Every effort is made to ensure samples are sterile however we recommend sterile filtering after dilution in media or the final working solution.
- Itoh, N. & Ohta, H. Fgf10: A Paracrine-Signaling Molecule in Development, Disease, and Regenerative Medicine. Curr. Mol. Med. 14, 504–509 (2014).
- Zhang, X. et al. Receptor specificity of the fibroblast growth factor family. The complete mammalian FGF family. J. Biol. Chem. 281, 15694–700 (2006).
- Igarashi, M., Finch, P. W. & Aaronson, S. A. Characterization of recombinant human fibroblast growth factor (FGF)-10 reveals functional similarities with keratinocyte growth factor (FGF-7). J. Biol. Chem. 273, 13230–5 (1998).
- Beer, H.-D. et al. The fibroblast growth factor binding protein is a novel interaction partner of FGF-7, FGF-10 and FGF-22 and regulates FGF activity: implications for epithelial repair. Oncogene 24, 5269–77 (2005).
- Ohuchi, H. et al. The mesenchymal factor, FGF10, initiates and maintains the outgrowth of the chick limb bud through interaction with FGF8, an apical ectodermal factor. Development 124, 2235–44 (1997).
- Min, H. et al. Fgf-10 is required for both limb and lung development and exhibits striking functional similarity to Drosophila branchless. Genes Dev. 12, 3156–61 (1998).
- Zhang, R.-R. et al. Stem Cell Reports Human iPSC-Derived Posterior Gut Progenitors Are Expandable and Capable of Forming Gut and Liver Organoids. Stem Cell Reports 10, 780–793 (2018).
- McCracken, K. W. et al. Modelling human development and disease in pluripotent stem-cell-derived gastric organoids. Nature 516, 400–404 (2014).
Fibroblast Growth Factor-10, FGFA, KGF-2, Keratinocyte growth factor 2
Result: FGF10 migrates as a single band at 17 kDa in non-reducing (NR) and upon reduction (R). No contaminating protein bands are visible.
Purified recombinant protein (7 µg) was resolved using 15% w/v SDS-PAGE in reduced (+β-mercaptothanol, R) and non-reduced conditions (NR) and stained with Coomassie Brilliant Blue R250.
Result: FGF10 supports proliferation and promotes epithelial to mesenchymal transition in human primary keratinocytes. Data and evaluation by Stemnovate Ltd.
Epithelial to mesenchymal transition (EMT) is a crucial morphogenetic process during development in which cells lose their epithelial characteristics and acquire migratory mesenchymal properties. FGF10 has an important role both during the embryonic EMT (type I) and on cancer cell initiation of metastasis (type III EMT).
Cell proliferation assays to assess the effect of Qkine FGF10 (0-100 ng/ml) on human primary epidermal keratinocytes in serum-free keratinocyte media. Cells were evaluated at culture days: 0 (baseline), 1, 2, 3, 4 days, as summarized schematically in Figure 1a. Figure 1b shows cell proliferation (Relative Luminescence Unit [RFU]) for days 1, 2, 3, 4 and normalized to day 0 readouts (n=3; P*<0.05 vs control). The log concentration plot in Figure 1c shows percent cell proliferation normalized over untreated control (%) and to day 0 (baseline) after 4 days treatment (n=3; P*<0.05). The maximal cell proliferation was observed at ~10ng/ml FGF10 and a reduction in cell number/viability as observed at 100 ng/ml. Data provided by Stemnovate Ltd, Cambridge, UK.
Representative images showing human primary epidermal keratinocytes treated with Qkine FGF10 at (0-100 ng/ml) at d0 (baseline), d1, d2, d3 and d4.
Induction of EMT in human primary keratinocytes following treatment with hFGF10. Induction of EMT was evaluated using immunofluorescence staining to determine expression of the epithelial marker (Cytokeratin 14 [CK14]) and mensenchymal marker (α-Smooth Muscle Actin [αSMA]) in Human primary epidermal keratinocytes after 4 days treatment with Qk003 hFGF10 (0-100 ng/ml).
Result: calculated molecular mass of the FGF10 is 16912.4 Da. Result of the analysis: 16781.2 Da which is consistent with the calculated mass. There are no minor contaminants.
MALDI mass spectrometric analysis is used to confirm the molecular mass of the intact protein and to reveal any heterogeneity that would not be evident in SDS-PAGE analysis. The results are compared with calculated mass of the protein with the assumption that all the cysteines are disulphide-linked. The different peaks represent different charge states of the protein.
Result: UV spectrum shows full recovery of protein following aliquoting and lyophilization.
Absorbance at 280 nm: average 0.172
Recovered concentration: 0.172 cm-1 x 10 / 0.144 cm-1 mg ml-1 = 1.2 mg / ml
Recovery: 120% (>100% due to routine 10-20% over-fill of vials during aliquoting)
The sample was diluted 1:10 in 100 mM sodium phosphate pH 7.4 and the UV spectrum 340-220 nm measured in duplicate. Concentration was calculated using extinction coefficient at 280 nm
Result: Endotoxin level <0.005 EU/ug protein (below level of detection)
Stem cell cultures are sensitive to endotoxins1, which can be present in media, serum and as a contaminant on plasticware. We optimize our protein production processes to ensure the lowest possible levels of endotoxin contamination. Our endotoxin pass criteria are set at the industry leading <0.1 EU per ug protein and we aim for <0.01 EU per ug protein. Endotoxin levels in our proteins are determined by an external expert microbiological testing services provider.
1. A biological study establishing the endotoxin limit for in vitro proliferation of human mesenchymal stem cells (2017). Yusuke Nomura, Chie Fukui, Yuki Morishita, Yuji Haishima. Regenerative Therapy, 7, 45-51.
View full batch quality testing data for Qk003
All our proteins are produced in our Cambridge, UK, labs. We provide detailed quality data for each batch because we believe reliable, high quality cytokines and growth factors are critical for successful stem cell and organoid culture.
When we test our proteins, we choose a vial at random and reconstitute as recommended to ensure we are testing as close to the protein you will receive as possible. Biochemical identity and purity is checked using SDS-PAGE, mass spectrometry and analytical reverse phase chromatography. Bioactivity is determined using an appropriate cell-based assay. As stem cells are sensitive to endotoxin levels, we use a high resolution test to ensure endotoxin levels are at industry leading low levels (<0.01 EU per µg protein). We also check that the correct amount of protein is recovered from the vial – it might sound basic but if you order 100 µg, we believe you should receive 100 µg so when you use the proteins you can rely on your calculated dilution.
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