Nupage sds page

Detection of dysferlin was performed using the following protocol with primary anti-dysferlin antibodies (NCL-Hamlet 2, Cliniscience) diluted 1:300 or (NCL-Hamlet, Abcam) diluted 1:200 on untreated and treated patient cell protein extracts. Immunoblots were done using a whole protein extract sample prepared in lysis buffer (100 mM Tris buffer at pH 6.8, 4% SDS, 10% glycerol and 1% 2-mercaptoethanol). A 1% reducing agent (Life Technologies) was added and the samples were boiled for ten minutes. 20 μg of sample was loaded onto each lane of a 3–8% NuPage SDS–PAGE (Life Technologies). Proteins were then separated under electrophoresis at 80 volts at room temperature. Proteins were finally transferred onto nitrocellulose membranes (at 260 mA for 2 h at 4°C). Membranes were incubated with blocking buffer (1% non-fat milk powder in TBS-T) for one hour at room temperature. Primary antibodies were then diluted in blocking buffer and incubated with the membranes at room temperature for 1 h, with constant agitation. After washing in TBS-T, membranes were then incubated with the relevant secondary antibodies, which were diluted 1:10,000 in blocking buffer, for one hour at room temperature. The membranes were finally washed in TBS-T and developed using Pico Chemiluminescent Substrate (Pierce). GAPDH (Millipore) was detected using a dilution of 1:10,000.