Recombinant human HGF (NK1) protein is a high purity NK1 isoform of human hepatocyte growth factor (HGF) that promotes efficient differentiation of human iPSCs to hepatocyte-like cells at just 10 ng/ml with highly homogeneous expression of the hepatic marker, HNF4α. The highly scalable animal-free manufacture and enhanced bioactivity make this suitable for chemically-defined media and reproducible scale-up.
Recombinant human HGF (NK1) protein (Qk013)
£93.00 – £696.00
- Purity & bioactivity data
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- Resuspension & storage
For final purity and activity tests on our proteins, we choose a vial at random and reconstitute as recommended. Biochemical identity and purity is determined using SDS-PAGE, mass spectrometry and analytical reversed-phase chromatography. Bioactivity is quantified 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.
Result: HGF NK1 migrates as major band at 20 kDa in non-reducing conditions and 18 kDa upon reduction.
Purified recombinant protein (7 µg) was resolved using 15% w/v SDS-PAGE in reduced (+DTT, R) and non-reduced conditions (NR) and stained with Coomassie Brilliant Blue R250. NB reduced samples were not boiled as the protein is sensitive to high temperatures, which causes degradation.
Bioactivity: Luciferase reporter assay
Result: HGF (NK1) activity is determined using the Promega serum response element luciferase reporter assay (# E1340) in transfected HEK293T cells.
Comparative bioactivity of Qk013 HGF (NK1) and a mammalian cell-expressed full-length HGF from another commercial supplier was demonstrated using a quantitative luciferase reporter assay. EC50 of 49.6 pM and 68.4 pM respectively. Cells are treated in duplicate with a serial dilution of HGF for 6 hours. Firefly luciferase activity is measured and normalized to the control Renilla luciferase activity.
Purity: Mass spec analysis
Result: Consistent with the calculated mass. No heterogeneity is present.
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 disulfide-linked. The different peaks represent different charge states of the protein.
Purity: Analytical reversed-phase chromatography
Result: Reversed-phase chromatogram shows single sharp peak showing that the protein is pure and homogeneous.
Purity: Endotoxin level determination
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.
High purity, animal and carrier protein-free HGF protein for chemically-defined media and more reproducible scale-up
Recombinant human HGF (NK1) protein promotes efficient differentiation of human iPSCs to hepatocyte-like cells
HGF (NK1) supports human iPSC differentiation to hepatocyte-like cells. Defined media was supplemented with Qk013 HGF (NK1) during day 8-18 of the hepatocyte differentiation protocol (adapted from Heck W, et al. 2015). Cells attain characteristic hexagonal epithelial morphology. Expression of hepatic marker, HNF4α, and polarisation marker, E-Cadherin (ECAD), was detected using immunofluorescence.
Highly homogeneous expression of HNF4α in hepatocyte-like cells differentiated from iPSCs using Qk013 HGF (NK1)
Growth in media supplemented with Qk013 HGF (NK1) leads to highly homogeneous expression of hepatic marker, HNF4α. iPSCs were treated with hepatocyte differentiation media supplemented with Qk013 HGF (NK1) or full-length, mammalian cell-derived HGF from another commercial source.
All experiments have been conducted by the specialist stem cell biotechnology company, Stemnovate Limited, in Cambridge, UK.
Our recombinant human HGF (NK1) protein may be poorly soluble in physiological solutions. Please follow the handling guidance for lyophilized cytokines below to minimize loss of protein due to precipitation or adsorption to plastic. We advise storing the recombinant protein at very low pH before dilution in cell culture media or final working solutions. Low pH will also assist in maintaining the correct disulfide structure of the protein by minimizing disulfide bond exchange reactions.
- Resuspension in physiological buffers may cause precipitation of stock solutions, hence we recommend dissolving our lyophilized cytokines in 10 mM HCl (1:1000 dilution of concentrated HCl) while keeping the protein concentration at 50 µg/ml or above, in order to avoid loss by adsorption to plasticware.
- To ensure you recover all of the protein, let the sample sit for a few minutes with the solubilization buffer at room temperature and pipette gently up and down (avoid foaming).
- Rinse the tube with some more 10 mM HCl and pool with the rest.
- The protein is tolerant of some freeze and thaw cycles, but as always with proteins, it is better to aliquot and store frozen.
- Our proteins 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.
- Store at -80°C for long-term storage, -20°C for short-term storage.
We 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.
Recovery: protein quantitation
Result: UV spectrum shows full recovery of protein following aliquoting and lyophilization.
Absorbance at 280 nm: average 0.144
Recovered concentration:0.144 cm-1 x 10 /1.272 cm-1 mg ml-1 = 1.13 mg / ml
Recovery: 113% (>100% due to routine 10% 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.