Ettan dige

Samples were compared in one experiment using 2-D DIGE (Ettan DIGE, Ge Healthcare, UK) as described in Di Luca et al., [15 (link)]. Samples used were muscle exudate collected from each of the three quality classes, HDrip, LDrip and IP (with four animals per class), at days 1, 3 and 7 post mortem; resulting in a total of 36 exudate samples. Using the minimal labelling technique [22 (link)], samples and internal standard were respectively labelled with Cy5 and Cy3 dye fluors (GE Healthcare, UK), according to the manufacturer’s instructions. Passive in-gel rehydration using immobilised DryStrips pH 4–7, 24 cm (GE Healthcare, UK) gradients [15 (link)], in which were loaded 50 μg of labelled sample proteins plus 50 μg of labelled pool proteins, was carried out overnight in the dark. The isoelectric focusing was performed using Ettan IPG Phor3 (GE Healthcare, UK) under the following conditions: 3500 V at 75000VHrs; gradient 8000 V for 10 min; 8000 V for 1Hour and holding step at 100 V. After isoelectric focusing IPG strips were reduced and then alkylated [15 (link)]. The proteins were further separated in the second dimension using a 12% SDS-PAGE gel at 15°C overnight in the dark by means of a PROTEAN Plus Dodeca Cell (Bio-Rad, Hercules, CA).