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 reverse 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 activity is determined using the Promega serum response element luciferase reporter assay (# E1340) in transfected HEK293T cells. EC50 = 0.70 ng/ml.
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. Data are from Qk013 batch #010.
Purity: mass spec analysis
Purity: analytical reverse phase chromatography
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.
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.
HGF NK1 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 to before dilution in cell culture media or final working solutions. Low pH will also assist in maintaining the correct disulphide structure of the protein by minimizing disulphide 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 stored frozen.
- 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.
- Store in -80°C for long term storage. -20°C for short-term.
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
View full batch quality testing data for Qk013
Batch #010, #011