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Then 0.75 ml of the instrument matrix solution (saturated solution of a -cyano-4- hydroxycinnamic acid in 50% ACN and 0.5% v/v trifluoroacetic acid) was applied to dried samples and dried again. Each protein spot digest (0.75 ml) was spotted into the MALDI instrument target and allowed to dry. Tryptic digestion was carried out overnight at 37 u C. The spots were excised and delivered into 96-well microplates where they were destained and dehydrated with acetonitrile (ACN) for subsequent rehydration with trypsin solution. A spot-picking list was generated and exported to Ettan Spot Picker (GE Healthcare). For 2-DE/Western blot analysis, gels (containing 80 m g of protein) were transferred onto a nitrocellulose membrane (0.8 mA/cm 2 1 h, 40 min) with a Pharmacia Biotech Nova Blot semi-dry transfer instrument. Each sample was carried out in triplicate under the same conditions. IPG strips were embedded at the top of a 1.5 mm thick vertical polyacrylamide gradient gel (8–16%T) using 0.5% w/v agarose and run at a constant current of 40 mA/gel at 20 u C. The second dimension was performed on an EttanDalt Six Electrophoresis system (GE Healthcare). The strips were first equilibrated with a buffer containing 50 mM Tris-HCl, pH 8.8, 6 M urea, 2% w/v SDS, 30% v/v glycerol, and 1% w/v DTT for 15 min then they were equilibrated again with the same buffer described above, except it contained 4% w/v iodoacetamide instead of DTT. Isoelectric focusing (IEF) was carried out for a total of 32 kV h. The samples were then combined with solubilizing buffer containing 8 M urea, 2% of 3-1-propane sulfonate (CHAPS), 0.3% DTT, 2% immobilized pH gradient (IPG) buffer, and a trace of bromophenol blue and loaded into 18 cm IPG strips 3–10 non linear on an Ettan IPGphor (GE Healthcare) apparatus system and rehydrated for 7 h. Samples (60 m g of protein determined using the Bradford method), were denatured a solution containing 10% of SDS, 2.3% of dithiothreitol (DTT) heated to 95 u C for 5 min. Two-Dimensional gel electrophoresis (2-DE) was used to separate ghost proteins as previous described. Cell signal semiquantitative analysis and cells 3D visualization were carried out with ImageJ an open source program (/ij). After three washes in PBS, fluorescent RBCs cells, mounted with a drop antifade, were visualized by confocal microscopy LSM-700 (Zeiss, Jena, Germany). A-11008 Alexa Fluor 488,) diluted 1:100 in PBS for 1 h at RT. Following washed in TBS, cells were incubated with specific secondary antibody goat anti-rabbit (cod. Cells were blocked with 1% BSA for 30 min at RT and incubated overnight at 4 u C with a monoclonal rabbit beta-actin antibody (Millipore Corporation, Billerica, MA, USA) diluted 1:50 in PBS. Erythrocytes purified as described before were smeared onto slides and after drying were fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) at RT for 10 min, permeabilized with 0.1% Triton X-100 in PBS for 5 min at 4 u C and washed three times in PBS. Optical densities were quantified using a computerized imaging system (Quantity One Imaging system).

Images were digitized (ChemiDoc XRS, BioRad, Hercules, CA). Following washed in TBS Tween, membranes were incubated with specific secondary antibodies for 1 h at RT and, finally, with ECL reagent (BioRad, Hercules, CA, USA). AB5605 Millipore Corporation, Billerica, MA, USA). actin and 2-DE RBC ghosts were performed using goat 4-HNE antibody (cod.