Motorneurondisease treatments a step closer uq_news MolPsychiatry
Mouse rostral cortices or lumbar spinal cords were collected for IB as described above and snap-frozen on dry ice. Tissues were then thawed on ice and homogenised in 5× v/w RIPA lysis buffer containing 1 mM PMSF and protease and phosphatase inhibitor cocktails with three 1.4 mm Zirconium oxide beads using Precellys tissue homogeniser . Samples were centrifuged at 4 °C, 100,000for 30 min, and the supernatant was taken as the RIPA-soluble fraction.
Protein samples of RIPA-soluble or RIPA-insoluble/urea-soluble fractions were separated by electrophoresis on a polyacrylamide gel containing 12% acrylamide in the presence of reducing agent . After SDS-PAGE, proteins were transferred to nitrocellulose membranes and incubated in blocking solution BSA, 0.05% Tween-20 in TBS ) and then incubated overnight in primary antibody diluted in the blocking solution.
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