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: FGF2 migrates as major band at 17 kDa in non-reducing (-βME) conditions and upon reduction (+βME).
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.
Bioactivity: luciferase reporter assay
Result: FGF2 activity is determined using the Promega serum response element luciferase reporter assay (*) in transfected HEK293T cells. EC50 = 0.2 ng/ml.
Cells are treated in duplicate with a serial dilution of FGF2 for 6 hours. Firefly luciferase activity is measured and normalized to the control Renilla luciferase activity. Data are from Qk027 batch #010
Purity: mass spec analysis
Result: calculated molecular mass of FGF2 is 17222 Da. Molecular mass from this analysis is 17222.5 Da, consistent with the calculated mass.
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: 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.
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.
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 0.119
Recovered concentration:0.9 cm-1 x 10 /0.93 cm-1 mg ml-1 = 0.96 mg / ml
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 Qk025
Batch #011, #012