The proliferation of NIH/3T3 cells treated with increasing concentrations of mEGF was assessed. After 24 hr treatment, cells were labeled with BrdU for 4 hr. BrdU incorporation was determined using the BrdU Cell Proliferation Assay Kit #6813 and the OD450 was determined.
The purity of recombinant mEGF was determined by SDS-PAGE of 6 µg reduced (+) and non-reduced (-) recombinant mEGF and staining overnight with Coomassie Blue.
Western blot analysis of extracts from NIH/3T3 cells, untreated or treated with mEGF for 10 minutes, using Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP® Rabbit mAb #4370 (upper) or p44/42 MAPK (Erk1/2) (137F5) Rabbit mAb #4695 (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 mEGF. 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 mEGF. All lots are greater than 98% pure.
Recombinant mEGF has a Met on the amino terminus and has a calculated MW of 6045. DTT-reduced and non-reduced protein migrate as 6 kDa polypeptides. The expected amino-terminal MNSYP of recombinant mEGF was verified by amino acid sequencing.
The bioactivity of recombinant mEGF was determined in an NIH/3T3 cell proliferation assay. The ED50 of each lot is between 10-250 pg/ml.
Less than 0.01 ng endotoxin/1 μg mEGF.
Recombinant mouse EGF (mEGF) Asn977-Arg1029 (Accession #NP_034243) was produced in E. coli at Cell Signaling Technology.
EGF is produced by epithelial cells, fibroblasts, and many other cell types (1,2). Low molecular weight soluble EGF is generated through proteolysis of a larger ~130,000 molecular weight transmembrane precursor (1,2). Both soluble and membrane forms of EGF are active (2). EGF induces proliferation, differentiation, and survival of many cell types including tumor-derived cells (1-3). There are multiple members of the EGF family and multiple members of the ErbB/HER EGF receptor family. EGF binds to ErbB1/HER1 and induces homodimerization or induces heterodimerization with other ErbB/HER members (1). Binding of EGF signals through the MAPK, PI3K/Akt, and Stat5 pathways (1). EGF, EGF family members, EGF receptors, and their signaling pathways are involved in many cancers and are targets for therapeutic intervention (1, 2).
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