Osteosarcoma and soft callus tissues were lysed in radioimmunoprecipitation assay buffer supplemented with 1% protease inhibitor (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), homogenized and incubated on ice for 30 min. The supernatant was collected by centrifugation at 15,000 × g at 4°C for 20 min. Crude tissue lysates were further prepared for 2DE analysis using a 2-D Clean-Up kit, according to the manufacturer's protocol (GE Healthcare Life Sciences, Uppsala, Sweden). Protein concentration was determined using Bradford assay. The protein pellets were resuspended in 2D lysis buffer. Individual samples (60 µg protein) were applied by overnight in-gel rehydration of 7 cm pH 3-10 nonlinear gradient IPG strips (GE Healthcare, Chicago, IL, USA). IEF was performed at 7,000 Vh, 55 mA per gel strip using an Ettan IPGphor 3 (GE Healthcare). The IPG strips were equilibrated and separated in 14% SDS-PAGE followed by SYPRO Ruby staining (Thermo Fisher Scientific, Inc., Waltham, MA, USA), as previously described (16 (link)). Gels were scanned and visualized using Ettan DIGE Imager (GE Healthcare).
Nonlinear gradient ipg strips
Manufactured by GE Healthcare
Sourced in United States, Italy
Nonlinear gradient IPG strips are a type of laboratory equipment used in the field of proteomics. They are designed to create a nonlinear pH gradient across the surface of the strip, which is used in the first dimension of two-dimensional gel electrophoresis (2D-PAGE) for the separation and analysis of complex protein mixtures.
Automatically generated - may contain errors
Lab products found in correlation
Sypro ruby staining, by Thermo Fisher Scientific (1 mentions)
Ettan ipgphor 3, by GE Healthcare (1 mentions)
Ettan dige imager, by GE Healthcare (1 mentions)
2 d clean up kit, by GE Healthcare (1 mentions)
Proteosilver plus silver stain kit, by Merck Group (1 mentions)
Ettan ipgphor 2 system, by GE Healthcare (1 mentions)
Ettan daltsix electrophoresis unit, by GE Healthcare (1 mentions)
2 protocols using nonlinear gradient ipg strips
1
2DE Analysis of Osteosarcoma Tissues
2
Fibroblast Proteomics Sample Preparation
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The protocol of the fibroblast preparation for the following proteomics analysis consisted firstly in the hSDFs homogenization by a pestle in 400 µL of ice-cold buffer (Tris-HCl pH 7.5; EDTA; EGTA; fenilmetilsulfonilfluoride in ethanol; protease inhibitor cocktail; Triton X-100) and the subsequent centrifugation at 13,000 rpm for 10 min at 4 °C. The proteins contained in the supernatant were quantified by the BCA (bicinchoninic acid) method in spectrophotometry (l=562 nm) using the BSA (bovine serum albumin) as standard. The glycerol was then added to the homogenate which was frozen to -80 °C. 150 µg of proteins for each sample were precipitated using a chloroform/methanol/water mixture (4:1:3 v/v) and resolubilized in an adequate buffer. The first dimension (1-DE) was achieved using 18 cm of pH 3-10 non-linear gradient IPG strips (GE Healthcare, Milan, Italy) and an Ettan IPGphor II system (GE Healthcare, USA). The strips were then loaded onto a 12% acrylamide gel (24 cm length, 1 mm thickness) and run in an Ettan DALTsix electrophoresis unit (GE Healthcare). The gels were dyed with silver stain (ProteoSilver Plus Silver Stain Kit; Sigma Aldrich).
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