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== 阶段四 抗体孵育染色 == *'''转膜后于5%脱脂奶(2 g脱脂奶粉加40 mlTBST)中封闭2 h(37℃，80 rmp），每一格加入5 ml 5%脱脂奶''' *'''TBST洗5 min×5次''' *'''一抗用3%脱脂奶(1.5 g脱脂奶加50 mlTBST)稀释(1格4-5 ml)''' *'''37℃孵育1 h并4℃过夜或37℃ 2 h,回收一抗，保存在-20℃,每次用前加一点(YAP 1 ul)''' *'''TBST洗5 min×5''' *'''二抗用3%脱脂奶(1.5 g脱脂奶加50 mlTBST)''' *'''37℃孵育1 h,TBST洗5 min×5''' *'''显色：葆光Millipore Immobilon western 显色=黑色：白色=1：1，一般各加1 ml，配成2 ml显影液'''
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