Ettan dige scanner

Prepared secreted proteins were separated by 2D-DIGE as described previously [49 (link)]. The Cy2, Cy3, Cy5 labeled samples (50 μg) were mixed and loaded on the strips (linear, 24 cm, pI 4–7, GE Healthcare, USA) for the first dimension separation. Next, the strips were placed on top of 12.5 % SDS-PAGE gels for the second dimension electrophoresis. Protein spots on gels were scanned using an Ettan DIGE Scanner (GE Healthcare, USA) and the images were analyzed using Decyder 2D software (Version 7.0, GE Healthcare, USA). Finally, spots from different gels were matched using Biological Variation Analysis. Only spots present in all gels and which exhibited statistically significant changes in intensity (≥1.5 fold or ≤ −1.5 fold, p <0.05) were considered to be differentially expressed proteins.

Samples were separated by SDS-PAGE and scanned with Cy3 filters to detect the TAMRA fluorophore using an Ettan DIGE scanner (GE Healthcare).
For western blot, proteins were transferred from gels to polyvinylidene fluoride membrane (Immobilon-P^SQ, Millipore) using a semidry system (Invitrogen). Following blocking (5% milk in Tris-buffered saline, 1% Tween), membranes were incubated with primary antibody for 1 hr at RT or overnight at 4°C in blocking solution, then incubated with secondary antibody (goat anti-rabbit IgG-HRP, Invitrogen, 1:10,000) for 1 hr in blocking solution. Detection was carried out using Luminata Crescendo Western HRP substrate (Millipore) according to the manufacturer’s instructions and on a Fujifilm LAS 3000 imager. Primary antibodies, LdHASPB (rabbit; ab 2-AE) (Alce et al., 1999 (link)), LmHASPB (rabbit, 336) (Alce et al., 1999 (link)), GP63 (rabbit, polyclonal; provided by R. McMaster, University of British Columbia) (Frommel et al., 1990 (link)), GFP (mouse, Santa Cruz), were used at 1:1,000–2,000.

Prepared secreted proteins were separated by 2D-DIGE as described previously [34 (link)]. The Cy2, Cy3 and Cy5 labeled samples (50 μg) were mixed and loaded on the strips (linear, 24cm, pI 4–7, GE Healthcare, USA) for the first dimension separation. Next, the strips were placed on top of 12.5% SDS-PAGE gels for the second dimension electrophoresis. Protein spots on gels were scanned using an Ettan DIGE Scanner (GE Healthcare, USA) and the images were analyzed using Decyder 2D software (Version 7.0, GE Healthcare, USA). Finally, spots from different gels were matched using Biological Variation Analysis. Only spots present in all gels and which exhibited statistically significant changes in intensity (≥1.5 fold or ≤-1.5 fold, p <0.05) were considered to be differentially expressed proteins.