Bone morphogenetic protein 2 (BMP-2) is a member of the BMP subgroup of the transforming growth factor beta (TGF-β) superfamily. It plays numerous roles in the developing embryo, such as embryonic patterning along the dorso-ventral axis, organogenesis, limb bud formation, and bone and cartilage growth. BMP2 is a potent differentiation factor and directs human pluripotent stem cells towards extra-embryonic endoderm, mesenchymal and neural lineages, and chondrocytes 1. BMP2 induces bone and cartilage formation in vitro and chondrogenesis in human adult mesenchymal stem cells2.
BMP2, like all other members in the TGF-β superfamily, is synthesised as a preproprotein consisting N-terminal signal peptide, 259 amino acid residue pro-domain and 114 residue mature domain. Proteolytic removal of the propeptide enables mature BMP2 to form active disulphide-linked homodimers, and heterodimers with BMP7.
Mature human BMP2 shares 100% aa sequence identity with mouse and rat BMP2 and 85% identity with the related protein, BMP-4 and less than 51% with other BMPs.
Summary: Qk007 mature domain of human BMP2 (Uniprot: P12643) expressed in E.coli and purified to homogeneity. Mature active protein is a disulphide-linked dimer.
Form: protein is provided lyophilised from a fully volatile solution without carrier protein.
Reconstitution instructions: BMP2 is very poorly soluble in physiological solutions. Please follow the handling guidance for lyophilised cytokines below to minimise 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 minimising disulphide bond exchange reactions.
- Resuspension in physiological buffers may cause precipitation of stock solutions, hence we recommend dissolving our lyophilised 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 solubilisation 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 minimise loss by adsorption.
- Store in -80°C for long term storage. -20°C for short-term.
Every effort is made to ensure samples are sterile however we recommend sterile filtering after dilution in media or the final working solution
Molecular mass: ~26 kDa (for the dimer)
- Pera, M. F. et al. Regulation of human embryonic stem cell differentiation by BMP-2 and its antagonist noggin. J. Cell Sci. 117, 1269–80 (2004).
- Schmitt, B. et al. BMP2 initiates chondrogenic lineage development of adult human mesenchymal stem cells in high-density culture. Differentiation. 71, 567–77 (2003).
Bone morphogenetic protein 2,BDA2, SSFSC, BMP-2A, BMP-2, Bone morphogenetic protein 2A
Result: BMP2 migrates as a single band at 26 kDa in non-reducing (NR) 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.
Result: BMP2 activity is determined using an 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 normalised to the control Renilla luciferase activity. Data are from QK007 batch #010.
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 disulphide-linked. The different peaks represent different charge states of the protein.
Result: Reverse phase chromatogram shows single sharp peak showing that the protein is pure and homogeneous.
Protein purity and structural homogeneity is analysed by reversed phase chromatography. 50 µg of protein, at 0.1 mg/ml in 10 mM HCl is analysed in ACE C4 4.6 x 250 mm column using 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.
Result: UV spectrum shows full recovery of protein following aliquoting and lyophilisation.
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. This was diluted 1:10 in 6 M guanidine hydrochloride, 20 mM sodium phosphate pH 7.4 and the UV spectrum 340-220 nm. 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 full batch quality testing data for Qk007
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 firstname.lastname@example.org 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). 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.