Criterion xt tris acetate precast gels

Purified phosphorylated CTD was mixed in 1X XT buffer (Bio-Rad) and run on 3–8% Criterion™ XT Tris-acetate Precast Gels (Bio-Rad) at 100 V until the dye front reached the end of the gel. Protein was then wet transferred to a 0.45 μm PVDF membrane (Millipore, IPVH00010) in ice-cold transfer buffer (25 mM Tris, 192 mM glycine, 10% methanol) at 250 mA for 2 hours at 4°C. After transfer, the membrane was blocked with 5% non-fat milk in TBS for 1 hour at room temperature, with shaking. The membrane was then incubated with a 1:5,000 dilution of anti-GFP (Abcam #ab290) antibodies in 5% non-fat milk in TBST overnight at 4°C, with shaking. The membrane was washed three times with TBST for 10 min at room temperature with shaking. The membrane was incubated with 1:10,000 secondary antibodies (GE health) for 1 hr at RT and washed three times in TBST for 5 mins. Membranes were developed with Femto ECL substrate (Thermo Scientific, 34095) and imaged using a CCD camera.