The production of TNF-α by MCF7 cells cultured with increasing concentrations of hLeptin was assessed. Cells were treated with hLeptin alone, boiled hLeptin, or hLeptin in the presence of a neutralizing antibody. Media from cells incubated with hLeptin for 24 hr was collected and assayed for TNF-α using the SignalKine™ Human TNF-α Sandwich ELISA Kit #7289 and the OD450 was determined.
The proliferation of leptin receptor-transfected BaF3 cells treated with increasing concentrations of hLeptin was assessed. After 48 hr treatment with hLeptin, cells were incubated with a tetrazolium salt and the OD450 - OD650 was determined.
The production of TNF-α by MCF7 cells cultured with increasing concentrations of hLeptin was assessed. Media from cells incubated with hLeptin for 24 hr was collected and assayed for TNF-α using the SignalKine™ Human TNF-α Sandwich ELISA Kit #7289 and the OD450 was determined.
The purity of recombinant hLeptin was determined by SDS-PAGE of 6 µg reduced (+) and non-reduced (-) recombinant hLeptin and staining overnight with Coomassie Blue.
With carrier: Lyophilized from a 0.22 μm filtered solution of PBS, pH 7.2 containing 20 μg BSA per 1 μg hLeptin. Carrier free: Lyophilized from a 0.22 μm filtered solution of PBS, pH 7.2.
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 hLeptin. All lots are greater than 98% pure.
Recombinant hLeptin has a Met on the amino terminus and has a calculated MW of 16,026. DTT-reduced and non-reduced protein migrate as 15 kDa polypeptides. The expected amino-terminal MVPIQ of recombinant hLeptin was verified by amino acid sequencing.
The bioactivity of recombinant hLeptin was determined by its ability to induce TNF-α production from MCF7 cells. The minimum concentration used to obtain this effect was 50 µg/ml.
Less than 0.01 ng endotoxin/1 μg hLeptin.
Recombinant human Leptin (hLeptin) Val22-Cys167 (Accession #NP_000221) was produced in E.coli at Cell Signaling Technology.
Leptin is a 16 kDa adipocyte-derived hormone that relays the status of the body’s energy reserves to the hypothalamus, resulting in the suppression of appetite and modulation of energy expenditure (1,2). Leptin functions as a pleiotropic cytokine with multiple roles in immune function and reproduction (1-4). There are at least five isoforms of the leptin receptor, but only OB-Rb is signaling competent (1,2). OB-Rb is expressed on a number of cells, including T cells, B cells, keratinocytes, and others (1-6). Leptin/OB-Rb interactions induce Jak2, PI3K, Erk1/2, and Stat3 activation (1,2). OB-Rb is often highly expressed in many tumor types, including breast, pancreatic, and Tumor Initiating Stem Cells (TISCS), providing a potential mechanistic link for the tumor promoting effects of obesity (2,7).
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