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 you can rely on your calculated dilution.
Result: BMP2 migrates as a single band at 26 kDa in non-reducing (NR) conditions and 13 kDa as a single monomeric species 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.
Bioactivity: luciferase reporter assay
Result: BMP2 activity is determined using a BMP2-responsive luciferase reporter assay in stably transfected HEK293T cells. EC50 = 0.30 nM.
Bioactivity is determined using a BMP2-responsive firefly luciferase reporter in stably transfected HEK293T cells. Cells are treated (in triplicate) with a serial dilution of BMP2 for 6 hours. Firefly luciferase activity is measured and normalized to the control Renilla luciferase activity. Data are from QK007 batch #010.
Purity: mass spec analysis
Result: calculated molecular mass of the BMP2 dimer is 26058 Da. Result of the analysis: 26057.5 Da which is consistent with the calculated mass. No significant 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.
Protein purity and structural homogeneity is analyzed by reversed-phase chromatography. 50 µg of protein, at 0.1 mg/ml in 10 mM HCl is analyzed in ACE C4 4.6 x 250 mm column eluted using a 10 – 90 % acetonitrile gradient in 0.1 % trifluoroacetic acid. Homogeneity is judged by the absence of multiple peaks and by the symmetry of the main peak. Blue line shows absorbance at 280 nm and the green line the acetonitrile gradient.
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. Example data from QK007 batch #010.
Pure recombinant BMP2 protein may be poorly soluble in physiological solutions. Please follow our handling guidance for lyophilized cytokines below to minimize loss of protein due to precipitation or adsorption to plastic. We advise storing our recombinant human BMP2 protein at very low pH before dilution in cell culture media or your final working solution. 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 or at -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 0.136
Recovered concentration: 0.136 cm-1 x 10 / 1.46 cm-1 mg ml-1 = 0.93 mg / ml
The sample was reconstituted in 10 mM HCl to a theoretical concentration of 1 mg/ml following instructions above. Concentration was calculated using extinction coefficient at 280 nm.