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Western blot有多种类型的膜可以用,不是所有类型的膜都适合做Stripping。 PVDF膜是最适合做Stripping的膜类型,蛋白样本的丢失量最小。

抗体清除液是什么?Western抗体清除液又称为一二抗清除液或膜再生液(stripping buffer),常用于WB中转移了蛋白的膜的重复利用。 A.用TBST一直洗膜,洗8hrs以上就可以清除多数蛋白的信号,信号过强的蛋白如内参除外。 所以,当你的抗体非常弱的时候,孵育过夜实际上相当于stripping,新的信号不会出现,原来的信号也会被漂洗干净,出现白板。 将膜放入剥离液中,浸泡30分钟,期间可轻轻晃动容器2 - 3次,以加速抗体的剥离过程。 剥离完成后,用TBST进行清洗,每次清洗3 - 5分钟,共清洗3次,以去除残留的剥离液和抗体。 小编推荐使用配方三,优点是可以常温使用,并且适用于NC和PVDF膜。 根据小编经验,配方三比较温和,亲测一张膜可以反复剥离和敷抗体三次没问题。

WB洗膜是去除非特异性结合,增加表达的特异性,而tbs和tbst都可以用来洗膜,但是tbst中的t是tween20,是离子去污剂。 推荐先整膜转封闭完之后再裁膜,可以有效避免把胶切得稀碎,胶是脆的,膜是软的,所以建议切膜不切胶。 整膜封闭还有一个好处是比较节约封闭液。 对于贴壁细胞: 4℃ 预冷的 PBS 洗 2~3 遍,用细胞刮子刮下细胞,或用胰蛋白酶处理细胞使细胞不再贴壁很紧,并用移液器吹打下细胞。 a.加入适量蒸馏水装有NC膜或 PVDF膜 的抗体孵育盒中,漂洗5min(液体量能没过膜即可) b.弃掉蒸馏水加入适量抗体洗脱液,摇床上漂洗5min;

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Western转膜之后,封闭之前不需要洗膜,直接放在封闭液里即可。 封闭后也不需要洗膜,直接加入一抗孵育。 如果需要染色检测胶中蛋白,可以在转膜前或转膜后用丽春红染色工作液进行染色,并用大量的水洗膜直至水变清无色。

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