Immobiline drystrips ph 3 10

Approximately, 50 µg samples of peptide digest were fractionated using an Agilent 3100 OFFGEL Fractionator (Agilent Technologies, Wilmington, DE) and 13 cm Immobiline Drystrips pH 3–10 (GE Healthcare BioSciences AB, Uppsala, Sweden, 17-6001-14). Fractionation was performed according to the Agilent instruction manual. Briefly, peptides were diluted in IPG buffer, pH 3–10 (GE Healthcare, 17-6000-87), containing 5% glycerol. 150 µl of peptide solution were loaded into each of 12 wells and focused for 20 kV hr with a maximum current of 50 µA and power of 200 mW (24–36 hr). Focused solutions were pipetted out of each well and the wells were re-extracted with 30% acetonitrile/0.1% TFA. Fractions 9 and 10 were combined, yielding 11 total fractions for subsequent LC-MS/MS analysis. Fractions were acidified with TFA, cleaned-up using stage tips, that is, pipette tips packed with reversed-phase membrane disks (Empore C-18 #2215, 3M Corporation, St Paul, MN), eluted with 60% acetonitrile, 0.1% TFA, and then concentrated in a Speed-Vac (Rappsilber et al., 2003 (link)).

Following the precipitation the pellet was solubilized with IEF buffer (8 M urea, 2% CHAPS, 100 mM DTT, 0.2% CA and 0.1% Bromophenol Blue). The isoelectric focusing was carried out in 7 cm Immobiline DryStrips pH 3–10 (GE Healthcare), under overnight rehydrating conditions with 200 μg protein in 150 μl IEF buffer. 12% polyacrylamide gels were used to separate proteins based on their molecular weight. 2D GE was made in three technical replicates. Because all of the 2D protein patterns from the different biological and technical replicates were identical, the final on-line nano-LC-MS/MS was made from three technical replicates 2D GE, and the spots were bulked.
After the 2D GE proteins were transferred to ImmobilonP PVDF membrane (Millipore, Billerica, USA) and blocked for 1 hour with 5% BSA and 0.05% TWEEN20 followed by an overnight incubation at 4 °C with 1:20 diluted blood sera. The immune-reactive proteins were detected with anti-Human IgA peroxidase- conjugated antibody produced in goat (Sigma-Aldrich-A0295) in the presence of 4-Chloro-1-Naphthol chromogenic peroxidase substrate. Immune responsive protein spots were excised and sent for protein identification using on-line nano-LC-MS/MS. Rice glutelin antibody coupled with anti-rabbit IgA as a secondary antibody was used in 2D Western blot analysis to confirm the presence of seed storage globulins14 (link).