Comparison of western blots processed manually vs. with the iBind Flex Western System. Samples containing GST-tagged recombinant proteins were separated on Invitrogen NuPAGE 4-12%, 20-well gels in MOPS SDS running buffer and then transferred to nitrocellulose membranes using the Invitrogen iBlot 2 Dry Blotting System. Blots were probed with identical concentrations of the same pair of primary and secondary antibodies. The primary antibody was rabbit anti-GST diluted 1:500 (8 µL in 4 mL Invitrogen iBind Flex Solution for the iBind system, 40 µL in 20 mL for manual tray incubation). The secondary antibody was goat anti-rabbit HRP diluted 1:600 (6.7 µL in 4 mL iBind Flex Solution for the iBind system, 33.3 µL in 20 mL for manual tray incubation). For final detection, blots were incubated for 5 minutes in Thermo Scientific SuperSignal West Dura Substrate for visualization with an imaging system.
Lanes 1-5: IKK beta (80 ng, 40 ng, 20 ng, 10 ng, 5 ng)
Lanes 6-10: DDR2 (120 ng, 60 ng, 30 ng, 15 ng, 7.5 ng)
Lanes 11-15: FLT1 (40 ng, 20 ng, 10 ng, 5 ng, 2.5 ng)
Lanes 16-20: HCK (360 ng, 180 ng, 90 ng, 45 ng, 22.5 ng)