【求助】关于western 酶标二抗问题

最新回复

• one (2014-5-30 15:30:29)

  我们实验室对多个牌子的脱脂奶粉进行了比较，得出结论，完达山的脱脂奶粉效果最好，背景很低，别的牌子的要差不少。现在实验室一直用它封闭，效果很好。
  二抗是从北京中杉公司买的进口分装的，0.1ml 130元，可2500倍稀释。

• yes4 (2014-5-30 15:30:50)

  一般封闭液的选择，如是用AP 二抗用脱脂奶粉，如用HRP 二抗用BSA ，具体为和如此，查下蛋白质技术或在版内搜下即知！

• 6327555 (2014-5-30 15:31:09)

  其实我也觉得用bsa应该没问题，可是我已经竭尽全力去优化封闭条件和洗膜时间都不行，无奈之下选择了询问我的酶标二抗公司(JACKSON IMMUNE)，以下是他们销售部经理回信：
  We do not recommend using BSA or dry milk because both contain small amounts of contaminating bovine IgG. Any anti-goat IgG cross-reacts very strongly with bovine IgG. When you block with BSA or dry milk, bovine IgG will stick to the blot everywhere and then you will label it with the anti-goat IgG. The whole blot will turn dark. For the same reason you should not dilute any anti-goat IgG with with buffer containing BSA or dry milk. The bovine IgG in the BSA or dry milk will bind to the anti-goat IgG and form immune complexes, which will lower the titer of the antibody. The same will happen with any anti-sheep IgG. For blocking a rabbit anti-goat IgG, it is best to use normal rabbit serum, but do not dilute antibodies with normal serum, just use it to block as the first step in the procedure. If you had a donkey anti-goat IgG, it is best to block with normal donkey serum, or if you had a mouse anti-goat IgG, it is best to block with normal mouse serum.
  Although it is common to use BSA or dry milk for blocking and diluting most antibodies, they may not be used with any anti-goat IgG or any anti-sheep IgG, because these antibody cross-react so strongly with bovine IgG. For this same reason care must be taken when labeling cells grown in tissue culture media containg fetal calf serum. Fetal calf serum contains IgG which may coat the surface of cells grown in the medium. When these cells are labeled with goat primaries and then anti-goat secondaries, there may be surface labeling of the cells just because of the bovine IgG.
  This is not a problem with most other antibodies, including anti-mouse IgG, anti-rabbit IgG, anti-rat, and others, especially those which have been adsorbed to eliminate cross-reaction with bovine IgG. Although it may be safe to block with BSA or dry milk in most cases, we think it is more effective to block with normal serum from the same species as the host species of the secondary antibody.
  主要意思就是当一抗为羊抗时因为羊和牛种属比较接近，任何抗羊的二抗交叉反映会很严重，自然背景会很深，他们推荐我用兔血清，也就是二抗同种属血清，可是我用了之后效果也不太好，艾，没办法，愁啊

• wmp1234 (2014-5-30 15:31:29)

  
  我也是觉得完达山的脱脂奶粉不错！

• 987789 (2014-5-30 15:31:51)

  我们实验室对多个牌子的脱脂奶粉进行了比较，得出结论，完达山的脱脂奶粉效果最好，背景很低，别的牌子的要差不少。现在实验室一直用它封闭，效果很好。
  
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  你说的别的牌子的奶粉是指国内的还是国外的？

• 6327555 (2014-5-30 15:32:15)

  可是我在沈阳转了好几个大型超市，都没有脱脂奶粉，只有低脂的，什么中兴、联营、沃尔马、华联、超市都转过了，没卖的，晕

• tuuu2 (2014-5-30 15:32:34)

  
  可以试试用酪蛋白来封闭膜。