Qk003 recombinant human FGF10

Protein quality information for Qk003 batch #010

Protein concentration is 1 mg/ml

Protein is provided in PBS without carrier protein

All our proteins are produced in-house by our scientists and we provide detailed quality data for each individual batch.  Please contact us any time by email support@qkine.com or phone +44 (0) 1223 491486 if you have any questions.

After aliquotting and lyophilising the protein, 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 of each batch is checked using SDS-PAGE, mass spectrometry and analytical reverse phase chromatography.  We use a sensitive test to ensure endotoxin levels are at industry leading low levels (<0.01 EU per µg protein). Bioactivity of the protein is determined using the quantitative activin responsive luciferase reporter assay.  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.

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 lyophilisation.

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 optimise 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 units sizes available and purchasing information online or email orders@qkine.com

Our products are for research use only and not for diagnostic or therapeutic use.  Products are not for resale.