Early diagnosis of radiodermatitis using lactate dehydrogenase isozymes in hairless mice (SKH1-hr)

Cho, Sung-Kyu; Kim, Won-Dong

Heart extracts from SKH1-hr mice were electrophoresed using native-PAGE and transferred to nitrocellulose membranes (Trans-Blot Transfer Medium, Bio-Rad Lab. Inc., Hercules, CA) at 25 V for 30 min by using the semi-dry transfer system (Trans-Blot SD cell, Bio-Rad Lab. Inc., Hercules, CA) in 15 mM Tris-glycine buffer (pH 8.3). Membranes were blocked with 5% skimmed milk in TBS (0.01 M Tris buffered saline, pH 7.5) for 1 hr and were then incubated with primary antibodies for 1 hr. LDH A 4 and B 4 isozymes were assayed using anti-LDH A 4 isozyme [22] and anti-B 4 isozyme [23] . After washing, the membranes were incubated with anti-rabbit IgG peroxidase-conjugated secondary antibodies for 1 hr and were then washed again. Immunodetection was performed using a chloronaphthol and hydrogen peroxide mixture.