Nupage bis tris gradient mini gels

Prior to PAGE, CSF samples were added to a mixture containing 4x LDS sample loading buffer (2% lithium dodecyl sulfate, 10% glycerol, 200 mM Tris; pH 8.4) and DTT (50 mM final concentration). The samples were diluted with PBS to ensure equal loading volumes and heated at 70°C for 10 minutes. Samples were run on 4-12% NuPAGE Bis-Tris gradient mini gels (Life Technologies; Grand Island, NY) at 150 V using MOPS buffer (50 mM MOPS, 50 mM Tris, 0.1% SDS, 1 mM EDTA; pH 7.7). Following completion of the PAGE run, samples were transferred to Immobilon FL PVDF membrane (Millipore; Darmstadt, Germany) using Towbin buffer (192 mM glycine, 25 mM Tris). To optimize the transfer of CSF high and low molecular weight proteins, we used a ramped overnight transfer strategy, previously shown to result in improved transfer of diverse molecular weight protein mixtures to membranes [42 (link)]. Membranes were transferred at a constant 8 V for 6 hours, then a constant 16 V for 6 hours. This ramped approach improved the transfer of CSF proteins as compared to common transfer strategies (e.g., 100 V for 1 hour, or 20 V for 12-16 hours; data not shown).