Hyperfilm mp x ray film

30 nM SPAK, 2 μM GST-NKCC1 (DU6146), ± 1 μM GST-MO25α (DU30906) were reacted at 30°C in a Thermomixer at 1000 rpm from times ranging from 0–20 min in ³²P radioactive kinase assay buffer (50 mM Tris/HCl pH 7.4, 0.1 mM EGTA, 10 mM MgCl₂, 0.1 mM [³²P]-ATP (~200 c.p.m/pmol), 1 μM ovalbumin) at a final volume of 20 μL. The reactions were quenched with 20 μL of 2x Laemmli sample buffer, heated to 70°C for 10 min. The samples were subjected to electrophoresis on polyacrylamide gels, and stained with Instant Blue™ for 1 hr. followed by destain in dH₂0 for 1 hr. The gels were rinsed in dH₂0 containing 5% glycerol and then sandwiched between two sheets of cellophane clamped to a gel-drying apparatus and dried in a GelAir Dryer (Bio-Rad Hercules, CA). Once dry, the gels were placed in an autoradiography cassette and exposed to GE Hyperfilm MP X-ray film overnight. The films were then developed using a Konica automatic developer. Following autoradiography, the bands corresponding to NKCC1 were excised from the dried gel, transferred to microcentrifuge tubes and ³²P-radioactivity incorporation was quantified by Cerenkov counting.