Umax powerlook 1120 scanner

Gels were scanned using a UMAX PowerLook 1120 scanner and Labscan software (GE Healthcare, Uppsala, Sweden) at 600 dpi. Images were analyzed using Image Master^TM 2D Platinum 6.0 software (GE Healthcare, Uppsala, Sweden). Spots were manually detected and analyzed using three independent sets of gels (sensitive MCF7 versus paclitaxel-resistant MCF7/PacR). The statistical significance of the change in expression of matched spots was determined using the Student´s t-test. Spots with at least a two-fold change in the signal were selected for MALDI-TOF mass spectrometry (MS) analysis.

Protein extraction was performed following the protocol described by Link et al. [106 (link)] to avoid vitellogenin sample contamination.
Protein quantification was carried out by densitometry analysis following SDS-PAGE. For that purpose, samples were run in precast gels (NuPAGE™ 4 to 12%, Bis-Tris, 1.0 mm, Mini Protein Gel, 10-well, Invitrogen). The LMW-SDS Marker Kit (GE Healthcare) was used as a standard. After staining with colloidal Coomassie blue, gel images were digitized with a UMAX Power-Look 1120 scanner and LabScan 5.0 software (GE Healthcare). Quantification was performed using ImageQuant TL software (v8.1), a 1D analysis module.
Twenty micrograms of protein extracted from each sample was run on a 3-cm-long SDS-PAGE gel. Each lane was excised into 3 fragments, transferred to Eppendorf tubes and processed independently. Proteins were in-gel reduced and alkylated by incubation with 10 mM DTT for 1 h at 56°C followed by incubation with 50 mM iodoacetamide for 45 min at room temperature. In-gel protein digestion was performed by incubation with 2 μg of trypsin (sequence grade, Promega) in 50 mM ammonium bicarbonate overnight at 37°C. Peptides were extracted from gel pieces by the addition of 60% acetonitrile/0.1% formic acid with vigorous agitation followed by vacuum drying and resuspension in 0.1% formic acid. Samples were centrifuged at 16,000xg for 30 min at 4°C.