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recombinant human/mouse/rat/bovine/porcine BMP-2 protein (Qk007)

Human/mouse/rat/bovine/porcine BMP2 protein (bone morphogenetic protein 2) protein is member of the TGFβ family and a key regulator of embryogenesis and potent differentiation factor of embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC) towards endoderm fates. BMP2 plays roles in the differentiation of mesenchymal cells to adipocytes, epithelial cancer EMT, chondrogenesis and regulation of neuronal and glial cell development.

26 kDa disulfide–linked bioactive highly pure dimer comprised of the mature domain of human BMP2 protein (animal-free and carrier protein-free). Our recombinant human BMP2 protein has been optimized for expression in E. coli and purified to homogeneity, offering a highly reliable and cost-effective reagent for the regulation of stem cell differentiation.

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25501005001000
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enquiries, bulk and stock reservation, please email orders@qkine.com

1mg may be despatched as 2 x 500µg

summary

  • high purity human/mouse/rat/bovine/porcine bone morphogenetic protein 2, BMP2 (Uniprot: P12643).

  • 26 kDa (dimer)

  • expressed in E. coli

  • animal-free (AOF) and carrier protein-free.

  • manufactured in our Cambridge, UK laboratories

  • lyophilized from acetonitrile, TFA

  • resuspend in 10mM HCl at >100 µg/ml, prepare single use aliquots, add carrier protein if desired and store frozen at -20 oC or -80 oC

handling and storage FAQ

featured applications

  • differentiation of human pluripotent stem cells towards extra-embryonic endoderm, mesenchymal, neural lineages, and chondrocytes

bioactivity

Human BMP2 Qk007 protein bioactivity lot #010

BMP2 activity Bioactivity is determined using a BMP2-responsive firefly luciferase reporter in stably transfected HEK293T cells. Cells are treated with a serial dilution of BMP2 for 6 hours. Firefly luciferase activity is measured and normalized to the control Renilla luciferase activity. EC50 = 0.3 nM. Data from lot #010. n=4

purity

Human BMP2 Qk007 protein purity SDS-PAGE lot #010

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. Data from Qk007 lot #010

further quality assays

  • mass spectrometry: single species with expected mass

  • analytical reversed-phase: single sharp peak

  • endotoxin: <0.005 EU/μg protein (below level of detection)

  • recovery from stock vial:  >95%

request batch-specific CoA

We are a company founded and run by scientists to provide a service and support innovation in stem cell biology and regenerative medicine.  All our products are exceptionally high purity, with complete characterisation and bioactivity analysis on every lot.

research areas

organoid
iPSC/ESC maintenance
neural
hepatocyte
pancreatic
endodermal
mesodermal
epithelial
mesenchymal
hematopoietic

protein background

Bone morphogenetic protein 2 (BMP2) 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 protein is a potent differentiation factor and directs human pluripotent stem cells towards extra-embryonic endoderm, mesenchymal and neural lineages, and chondrocytes1.  Recombinant BMP2 protein induces bone and cartilage formation in vitro and chondrogenesis in human adult mesenchymal stem cells2.

Human bone morphogenetic protein 2 (BMP2) is synthesized as a preproprotein consisting of N-terminal signal peptide, 259 amino acid residue pro-domain and 114 residue mature domain. Proteolytic removal of the propeptide enables mature BMP2 protein to form active disulfide-linked homodimers, and heterodimers with BMP7.

Mature human BMP2 protein shares 100% amino acid sequence identity with mouse and rat BMP2. It also shares 85% amino acid sequence identity with the related protein, BMP4, and less than 51% identity with other BMPs.

product documents

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.

Purity: SDS-PAGE

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.

Recombinant human BMP2 protein purity in SDS-PAGE

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.

Recombinant human BMP2 bioactivity in a BMP2-responsive luciferase reporter assay

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.

Recombinant human BMP2 protein shows the expected molecular mass in MALDI mass spectrometry analysis

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.

Recovery of recombinant human BMP2 protein

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.

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.

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
Recovery: 93%

Recovery of recombinant human BMP2 protein

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.

Our products are for research use only and not for diagnostic or therapeutic use.  Products are not for resale.

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