Ell lysates were lysed and scraped in the buffer mentioned above. > 자유게시판

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Ell lysates were lysed and scraped in the buffer mentioned above. Cell extracts were then centrifuged at 280 g for 30 min at 4 and supernatant collected for analysis by Western blot.Protein concentration measurementThe protein concentrations of membrane samples were determined using Bradford Assay kit (Bio-Rad, Hercules, CA) according to manufacturer's instructions. Subsequently, membrane proteins were precipitated with acetone for 90 min, centrifuged at 10,000 g for 20 min at room temperature. The protein pellet was air dried and then dissolved in labeling buffer (30 mM Tris/HCL, pH 8.1, 7 M urea, 2 M thiourea, 1 (w/v) C7BzO). The final protein concentration was adjusted to 5 g/l with labeling buffer, snap frozen on dry ice and stored at -80?C until use.Western PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/23324267 blot analysisTo investigate the purity of membrane isolation, Western blot analyses were performed. Both Membrane (M) and Cytosol (C) fraction extracts were resolved on 11 SDS-PAGE gels and transferred to nitrocellulose membranes. The same amount of protein (30 g) was loaded. All membranes were incubated with Ponceau-S (Sigma)Fluorescent labeling of proteins was performed using minimal CyDye labeling methodology as described by the manufacturer (GE Healthcare Bioscience, Uppsala, Sweden) and detailed elsewhere [16,17]. Briefly, labeling was carried out on ice in the dark using 200 pmol dye per 50 g protein at a protein concentration of 5 g/l for 30 min. The labeling reaction was quenched by the addition of 1/10 vol of 10 mM lysine. Samples were either labeled with Cy3 or Cy5 with an internal standard (comprised of equal amounts of protein from each sample) labeled with Cy2. On completion of labeling, the samples were combined as recommended by the manufacturer. The combined samples were then precipitated in acetone as described above and the dried protein Capecitabine pellet re-suspended in 450 l of the solubilisation buffer (40 mM Tris, 7 M urea, 2 M thiourea, 1 (w.v) C7BzO) prior to isoelectric focusing. Isoelectric focusing (IEF) was performed using 24 cm immobilized pH gradient strips (IPGs) covering the pH range of 3-10, Tracking Dye, DTT to 100 mM and ampholytes to 0.5 (v/v) were added and each sample loaded by passive rehydration for 6 h at RT [17]. IPG strips were focused overnight using an IPGphor instrument (GE Healthcare, BUCKS, UK), according to the following profile: current limited to 60 A per strip, 100 V for 90 min, 300 V for 90 min, 500 V for 3 h, gradient to 1000 V for 4 h, gradient to 8000 V for 3 h and then constant 8000 V overnight until reaching between 60 000 and 80 000 Vh. Following focusing, the strips were incubated in 10 ml of equilibration buffer (50 mM Tris/ HCL, pH 8.8, 6 M urea, 30 (v/v) glycerol, 2 (w/v) SDS, 0.01 (w/v) bromophenol blue) for 20 min on aYap et al. Reproductive Biology and Endocrinology 2011, 9:73 http://www.rbej.com/content/9/1/Page 4 ofshaking platform at 80 rpm. Second dimension PAGE was performed on 8-16 gradient polyacrylamide gels (24 ?24 cm) overnight in a BioRad Dodeca cell (Richmond, CA) at 50 V as described by the manufacturer. The run was terminated when the dye front had reached the bottom of the gel. 2D gels were scanned using a Fuji FLA5100 laser scanner (Fujifilm, Tokyo, Japan) at excitation wavelengths of 473, 535 and 635 nm and with specialty dual wavelength emission filters for Cy2, Cy3 and Cy5 respectively. Image alignment, spot detection, background removal and expression analysis were performed using PG240 SameSpots.