The proliferation of NIH/3T3 cells treated with increasing concentrations of hPDGF-AA was assessed. After 24 hr treatment, cells were labeled with BrdU for 4 hrs. BrdU incorporation was determined by ELISA and the OD450-OD650 was determined.
The purity of recombinant hPDGF-AA was determined by SDS-PAGE of 6 µg reduced (+) and non-reduced (-) recombinant hPDGF-AA and staining overnight with Coomassie Blue.
Western blot analysis of extracts from NIH/3T3 cells untreated or treated with hPDGF-AA for 10 minutes, using Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb #4060 (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-AA. 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-AA. All lots are greater than 98% pure.
Recombinant hPDGF-AA does not have a Met on the amino terminus and has a calculated MW of 14,305. DTT-reduced protein migrates as an 18 kDa polypeptide and the non-reduced cystine-linked homodimer migrates as a 34 kDa protein. The expected amino-terminal SIEEA of recombinant hPDGF-AA was verified by amino acid sequencing.
The bioactivity of recombinant hPDGF-AA was determined in a NIH/3T3 proliferation assay. The ED50 of each lot is between 4-12 ng/ml.
Less than 0.01 ng endotoxin/1 μg hPDGF-AA.
Recombinant human PDGF-AA (hPDGF-AA) Ser87-Thr211 (Accession #NP_002598) was produced in E. coli at Cell Signaling Technology.
PDGF-AA is integrally involved in embryonic development, angiogenesis and organogenesis and induces fibroblast proliferation and migration (1,2). PDGF-AA is produced by epithelial, muscle, osteosarcoma and neuronal progenitor cells (1,3). Active PDGF-AA is formed through intracellular proteolytic cleavage of a large precursor. PDGF-AA is also concentrated in the extracellular matrix through alternative splicing that generates an extended carboxy-terminal that binds components of the extracellular matrix. The carboxy-terminal stretch is removed extracellularly to generate mature PDGF-AA (1,2). PDGF-AA binding to the PDGFR-α activates the receptor tyrosine kinase (1). PDGF-AA-induced signaling is through the Ras-MAPK, PI3K/AKT and PLCγ pathways (1). Dysregulation of PDGF-AA expression and/or signaling is often associated with cancer and fibrotic disorders (1).
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