Membranes were imaged on an iBright Imaging System, with the same time and image adjustment settings for each blot. For fluorescence: multiplex detection, the membranes were probed overnight with a mixture of primary antibodies diluted in blocking solution: Rabbit-anti Vinculin (1:15,000), Rat anti-α Tubulin (1:5,000)), and Mouse-anti p23 (1:5,000) followed by an incubation with secondary antibodies diluted at 1:5,000 in 1X Blocker FL: Donkey anti-Rabbit Alexa Fluor Plus 680, Donkey anti-Rat Alexa Fluor 488, and Donkey anti-Mouse Alexa Fluor Plus 800 for 1 hour. Band loss and smearing can be seen at the higher loads for all targets on the Bio-Rad blot, while the blot for the NuPAGE Bis-Tris gel offers superior protein loading capacity above 24 µg and crisp, bright bands.įor immunodetection: membranes were blocked for 1 hour in 1X Blocker FL Fluorescent Blocking Buffer. Both membranes were probed for three targets (vinculin, α-tubulin, and p23) using fluorescent immunodetection. Western blots using NuPAGE 4-12% Bis-Tris midi gels generate sharper bands at greater protein and RIPA lysis buffer loads than Bio-Rad 4–20% TGX midi gels.A NuPAGE 4–12% Bis-Tris midi gel, 12+2 well, was loaded with decreasing total protein amount of HEK293 lysate, subjected to electrophoresis in a SureLock Tandem Midi Gel Tank and transferred onto a 0.45 µm PVDF membrane using the SureLock Tandem Blot Module and the Bio-Rad 4–20% TGX midi gel, 12+2 well, was subjected to electrophoresis in a Criterion Midi Cell Tank and transferred onto a 0.45 µm PVDF membrane using the Criterion Blotter. *Not all percentages are available in every well type WedgeWell format: 10, 12, 15 17 well (load up to 2X sample volume per well) SureLock Tandem Midi Gel Tank, Invitrogen XCell4 SureLock Midi-Cell or Bio-Rad Criterion (with adapters only) Mini Gel Tank or XCell SureLock Mini-Cell NuPAGE MES SDS or NuPAGE MOPS SDS Running Bufferīolt MES SDS or Bolt MOPS SDS running buffer Working with dilute samples or need to load larger sample volumes, or when faster run times are neededĬoefficient of variation (CV) of only 2% for Rf values (migration) Need flexibility in well and gel format, or when you need a higher-throughput midi gel for a larger number of samples
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