HepG2 cells (1 × 106) were seeded in 15 cm dishes and, once confluence was reached, were washed with FBS-free DMEM. Control, PEITC 12 μM, dasatinib 1.25 μM, and their combination were added to the plates in 30 mL FBS-free DMEM. The conditioned medium from each plate was collected and centrifuged at 1000× g 10 min to remove cells. The supernatant was collected and concentrated with a 0.45 µM filter and added to Amicon® Ultra-15 3 kDa Centrifugal Filter Unit (Merck Life Science, Gillingham, UK). Filter tubes were centrifuged at 4 °C for 3000× g at steps of 30 min until the volume reached 500 µL (~30× concentration). Samples were aliquoted, quantified using Bradford assay, and stored at −80 °C. Conditioned medium was then diluted with EGM2+ prior HUVECs treatment for different assays or subjected to analysis using the Proteome Profiler Human Angiogenesis Array Kit (Bio-Techne Ltd., Abingdon, UK).
Amicon ultra 15 3 kda centrifugal filter unit
Manufactured by Merck Group
Sourced in United Kingdom, Germany
The Amicon® Ultra-15 3 kDa Centrifugal Filter Unit is a laboratory tool used for sample concentration and buffer exchange. It features a regenerated cellulose membrane with a molecular weight cut-off of 3 kDa, designed to retain molecules larger than the cut-off while allowing smaller molecules to pass through.
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Lab products found in correlation
2 protocols using amicon ultra 15 3 kda centrifugal filter unit
1
Conditioned Medium Collection and Analysis
2
Protein Refolding and Purification Protocol
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The purified protein sample was dissolved in deionized water containing 6 M GdmCl, 0.1 M Na2HPO4, 20 mM TCEP at pH 7.0, to obtain a concentration of 1.5 mM. The mixture was shaken overnight at 25 °C. Salts were removed with a PD-10 desalting column (Sephadex G-25 M, GE Healthcare). The obtained product was lyophilized and subsequently dissolved in 10 mM HCl and slowly added to degassed buffer containing 0.1 M Tris, 0.2 M KCl, 1 mM EDTA, 1 M GdmCl, 150 μM GSSG and 300 μM GSH at pH 8.7 with a final protein concentration of 30 μM. The refolding reaction was carried out at 37 °C for 24 h. The sample was concentrated with Amicon® Ultra-15 3 kDa Centrifugal Filter Unit (Merck, Darmstadt, Germany), followed by lyophilization. Refolded protein was further purified by RP-HPLC with a linear gradient starting with 10% to 80% B over 18 min at a flowrate of 15 mL min−1. Purity of fractions was analyzed by analytical RP-HPLC and ESI-MS (SI).
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