Chemiluminescent peroxidase substrate 3

A purified polyclonal anti-OtFea1 antibody was raised in rabbit against the peptide CTKYEFPKTRASGNY by GenScript HK Limited. For immunoblot analysis, the cells were lysed in loading buffer (62.4 mM Tris/HCl, 2 % (w/v) sodium dodecyl sulfate, 10 % (w/v) glycerin, 0.01 % (w/v) bromophenol blue, pH 6.8). The lysates were boiled for 5 min, separated (10 μg protein/lane) on an 8 % polyacrylamide gel and electroblotted onto a nitrocellulose membrane. Following the blocking of nonspecific binding sites with 5 % (w/v) milk powder in PBS-T, the membranes were incubated at room temperature with anti-OtFea1 (1:2000 in 5 % (w/v) milk powder in PBS-T). The membranes were washed three times with 5 % (w/v) milk powder in PBS-T (15 min each) and then incubated for 1 h at room temperature with peroxidase-conjugated anti-rabbit-IgY antibody (1:20,000 in 5 % (w/v) milk powder in PBS-T). The membranes were washed three times with 5 % (w/v) milk powder in PBS-T (15 min each), washed three times with PBS-T (15 min each) and developed with Chemiluminescent Peroxidase Substrate-3 (Sigma-Aldrich).

Cells were lysed in Laemmli sample buffer and denatured at 100 °C for 5 min before separation on 10% SDS–PAGE and then electrotransferred onto nitrocellulose blotting membrane (Amersham Protran). After transfer, the membrane was saturated in DPBS containing 0.1% Tween 20 and 5% milk. Primary antibodies (appropriate dilution) were added overnight at 4 °C. After washes in the presence of DPBS, appropriate secondary antibodies coupled with peroxidase were added. Immunoblotting was revealed with chemiluminescent peroxidase substrate (Chemiluminescent Peroxidase Substrate-3; Sigma-Aldrich) and exposure was observed with G: box (Syngene).