The samples were solubilized in a reducing loading buffer (62.5 mM Tris, pH 6.8, 6 M urea, 10% glycerol, 2% sodium dodecyl sulfate (SDS), 0.003% bromophenol blue, 5% 2-mercaptoethanol), electrophoresed in 10% SDS polyacrylamide gels and then transferred onto PVDF membranes.
Another approach to reduce to loading time is the implemented binary export format the molecules can be saved together with their generated scaffolds and such data set can be opened without even being sent to the server, reducing loading time from minutes to seconds for the largest data sets.
Samples (20 µg protein) in reducing loading buffer were boiled and electrophoresed through 8% SDS-PAGE gels (Pierce, Rockford, IL).
The proteins were eluted from the beads by heating for 5 minute at 100°C with reducing loading buffer and run on an SDS PAGE.
The beads were then washed with lysis buffer and the immune-precipitated proteins were eluted in reducing loading buffer and analyzed by dual-wavelength 1D-PAGE using a Typhoon 9410 (Amersham, Uppsala, Sweden).
Samples were boiled for 5 min in reducing loading buffer before separation on SDS-PAGE gels.
