Human Bone Morphogenetic protein-2 Recombinant

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Human Bone Morphogenetic protein-2 Recombinant

$70.00$4,700.00


accession P12643


Source Optimized DNA sequence encoding HumanBone Morphogenetic Protein-2 mature chain was expressed in Escherichia Coli.
Molecular weight Native human Bone Morphogenetic Protein-2 isgenerated by the proteolytic removal of the signal peptide and propeptide, the molecule has a calculated molecular mass of approximately13 kDa. Recombinant BMP-2 is a disulfide-linked homodimeric protein consisting of two amino acid residue subunits.BMP2 migrates as an approximately25 kDa protein under non-reducing conditions and as a kDa protein under reducing conditions in SDS-PAGE.
Purity >95%, as determined by SDS-PAGE and HPLC
Biological Activity The ED(50) was determinedby its ability to induce alkaline phosphatase production by C2C12 myogenic cells cells was found to be less than ng/ml.

Protein Sequence MVAGTRCLLA LLLPQVLLGG AAGLVPELGR RKFAAASSGR PSSQPSDEVL SEFELRLLSM FGLKQRPTPS RDAVVPPYML DLYRRHSGQP GSPAPDHRLE RAASRANTVR SFHHEESLEE LPETSGKTTR RFFFNLSSIP TEEFITSAEL QVFREQMQDA LGNNSSFHHR INIYEIIKPA TANSKFPVTR LLDTRLVNQN ASRWESFDVT PAVMRWTAQG HANHGFVVEV AHLEEKQGVS KRHVRISRSL HQDEHSWSQI RPLLVTFGHD GKGHPLHKRE KRQAKHKQRK RLKSSCKRHP LYVDFSDVGW NDWIVAPPGY HAFYCHGECP FPLADHLNST NHAIVQTLVN SVNSKIPKAC CVPTELSAIS MLYLDENEKV VLKNYQDMVV EGCGCR
Endotoxin Endotoxin content was assayed using a LAL gel clot method. Endotoxin level was found to be less than.1 ng/µg(1EU/µg).
Presentation Recombinant Human Bone Morphogenetic Protein-2 was lyophilized from a.2 μm filtered solution inmM AcOH pH.5.
Reconstitution A quick spin of the vial followed by reconstitution in distilled water to a concentration not less than.1 mg/mL. This solution can then be diluted into other buffers.
Storage The lyophilized protein is stable for at least years from date of receipt at -20° C. Upon reconstitution, this cytokine can be stored in working aliquots at° -° C for one month, or at -20° C for six months, with a carrier protein without detectable loss of activity. Avoid repeated freeze/thaw cycles.
Usage This cytokine product is for research purposes only.It may not be used for therapeutics or diagnostic purposes.


Biological Process Chondrogenesis
Biological Process Differentiation
Biological Process Osteogenesis
Molecular function Cytokine
Molecular function Developmental-protein
Molecular function Growth-factor

Methods

Adsorption of growth factors to osteoblast-secreted ECMs

  • Recombinant human TGF-β1 and BMP-2 were reconstituted to a final concentration of 1 mg/ml in PBS containing bovine serum albumin (BSA) as a carrier.
  • The growth factors were fluorescently labeled using the Dylight 488 labeling kit as described by the manufacturer.
  • Briefly, the growth factors were incubated with the fluorescent dye for 1 h at room temperature.
  • The unincorporated dye was removed by passing the mix through a desalting spin column.
  • The labeled growth factors were diluted in PBS to obtain a range of concentrations from 0 to 200 ng/ml.
  • ECMs prepared as described above were incubated overnight at 4°C with 200 µl of PBS containing either TGF-β1 or BMP-2 at different concentrations.
  • After 24 h, the growth factor solution was collected, ECMs were rinsed twice with PBS to remove unbound growth factor, and the distribution of bound protein on ECMs was imaged using a Nikon Eclipse TE2000-U fluorescent microscope.
  • Growth factor-bound ECMs were then…
  • Recombinant human TGF-β1 and BMP-2 were reconstituted to a final concentration of 1 mg/ml in PBS containing bovine serum albumin (BSA) as a carrier.
  • The growth factors were fluorescently labeled using the Dylight 488 labeling kit as described by the manufacturer.
  • Briefly, the growth factors were incubated with the fluorescent dye for 1 h at room temperature.
  • The unincorporated dye was removed by passing the mix through a desalting spin column.
  • The labeled growth factors were diluted in PBS to obtain a range of concentrations from 0 to 200 ng/ml.
  • ECMs prepared as described above were incubated overnight at 4°C with 200 µl of PBS containing either TGF-β1 or BMP-2 at different concentrations.
  • After 24 h, the growth factor solution was collected, ECMs were rinsed twice with PBS to remove unbound growth factor, and the distribution of bound protein on ECMs was imaged using a Nikon Eclipse TE2000-U fluorescent microscope.
  • Growth factor-bound ECMs were then scraped in PBS (100 µl/well) using a cell scraper, and bound concentrations of each protein were quantified via fluorescence (excitation: 493 nm, emission: 518 nm) with a plate reader .
  • Growth factor concentration was calculated from a standard curve corrected for BSA.
  • All concentrations were normalized to ECM protein concentrations, and the dissociation constants (Kd) were calculated from a Scatchard plot analysis using Prism .

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Growth Factors

  • ecombinant human Noggin/Fc chimera and neutralizing anti-human /4 antibody from , and BMP-4 from.

Chondrogenic Differentiation

  • sfMPCs were plated in triplicate (100,000 cells/well/24 well dish) and exposed to chondrogenic media for 14 days with or without micro-mass aggregation.
  • Aggregation was achieved by placing 100,000 cells in a 1.5 ml sterile tube at 37°C overnight.
  • Differentiation media consisted of sMPC culture media with 500 ng/mL BMP-2 , 10 ng/mL TGF-β3 , 10−8 M dexamethasone , 50 µg/mL ascorbic acid , 40 µg/mL proline , 100 µg/mL pyruvate and supplemented with insulin, transferrin, and selenium .
  • Media was changed every three days during the 14-day differentiation period.

Chromatin immunoprecipitation (ChIP)

  • ChIP was performed using the EZ-ChIP kit.
  • The isolated calvaria cells were differentiated by treatment with 150 ng/ml rhBMP-2 for 2 days and harvested for analysis.
  • Immunoprecipitations were carried out using the mouse anti-FLAG antibody and primers for analysis were designed to amplify the osteocalcin binding element; forward primer 5′-CTGAACTGGGCAAATGAGGACA-3′, reverse primer 5′-AGGGGATGCTGCCAGGACTAAT-3′.

Micromass Mesodermal Cultures

  • The effect of BMP modulators and BMP2 were analyzed by adding recombinant protein to the medium in 24 hr cultures.
  • Treatments were maintained for another 24 hr period.
  • After testing different protein concentrations we selected the following: human recombinant BMP2 200 ngr/ml ; human recombinant NOGGIN, 200 ngr/ml ; human recombinant CHL-1, 2400 ngr/ml ; mouse recombinant CHL-2 1200 ngr/ml ; human recombinant TSG 1000 ngr/ml ; mouse recombinant AN 3000 ngr/ml ; Follistatin 800 ngr/ml .
  • After these treatments we analyzed by Q-PCR changes in the expression of cartilage markers (Sox9, type 2; and Bmpr1b), fibrogenic markers (, type 1 and ), and joint markers (Activinβα, Gdf5, and Jaws).
  • The selected genes are well known markers of the corresponding morpho-developmental processes.
  • Only Jaws has not been used very often as joint marker, but it has been shown that it is essential for the formation of interphalangeal joints