The proliferation of NIH/3T3 cells treated with increasing concentrations of hPDGF-BB basic was assessed. After 24 hr treatment, cells were labeled with BrdU for 4 hrs. BrdU incorporation was determined by ELISA and the OD450-OD690 was determined.
The purity of recombinant hPDGF-BB was determined by SDS-PAGE of 6 µg reduced (+) and non-reduced (-) recombinant hPDGF-BB and staining overnight with Coomassie Blue.
Western blot analysis of extracts from NIH/3T3 cells untreated or treated with hPDGF-BB for 10 minutes, using Phospho-Akt (Thr308) (C31E5E) Rabbit mAb #2965 (upper) and Akt1 (C73H10) Rabbit mAb #2938 (lower).
With carrier: Lyophilized from a 0.22 μm filtered solution of 20 mM citrate, pH 3.0 containing 100 mM NaCl and 20 μg BSA per 1 μg hPDGF-BB. Carrier free: Lyophilized from a 0.22 μm filtered solution of 20 mM citrate, pH 3.0 containing 100 mM NaCl.
Stable in lyophilized state at 4°C for 1 year after receipt. Sterile stock solutions reconstituted with carrier protein are stable at 4°C for 2 months and at -20°C for 6 months. Avoid repeated freeze-thaw cycles.Maintain sterility. Storage at -20°C should be in a manual defrost freezer.
>98% as determined by SDS-PAGE of 6 μg reduced (+) and non-reduced (-) recombinant hPDGF-BB. All lots are greater than 98% pure.
Recombinant hPDGF-BB does not have a Met on the amino terminus and has a calculated MW of 12,294. DTT-reduced protein migrates as a 14 kDa polypeptide and the non-reduced cystine-linked homodimer migrates as a 32 kDa protein. The expected amino-terminal SLGSL of recombinant hPDGF-BB was verified by amino acid sequencing.
The bioactivity of recombinant hPDGF-BB was determined in a NIH/3T3 cell proliferation assay. The ED50 of each lot is between 4.0 - 15 ng/ml.
Less than 0.01 ng endotoxin/1 μg hPDGF-BB.
Recombinant human PDGF-BB (hPDGF-BB) Ser82-Thr190 (Accession #NP_002599) was produced in E. coli at Cell Signaling Technology.
PDGF-BB is a cystine-linked homodimer PDGF family member with key roles in development, cell proliferation, cell survival, and angiogenesis (1,2). PDGF-BB is expressed by vascular endothelium, megakaryocytes and Leydig cells (2). PDGF-BB targets pericytes, fibroblasts, monocytes and other cell types (1-3). PDGF induces fibroblast growth and migration (3) and is a chemoattractant for monocytes and granulocytes. Precursor PDGF-BB is cleaved intracellularly to generate a form that contains a carboxy-terminal stretch that serves to retain PDGF-BB in the extracellular matrix. In a second cleavage event, the carboxy-terminal stretch is removed extracellularly to generate mature PDGF-BB (1,2). PDGF-BB binds to PDGFRβ and induces receptor dimerization. Signaling is through the PI3K/Akt, JNK, and PLCγ pathways (1, 2). PDGF-BB may have a role in some cancer types (2).
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