【求助】关于pull down 求救
丁香园论坛
6313
我最近在做pull down 实验，先用western blot 标记的是被拉的蛋白，结果片子上是一片白板，连作为对照的input（只有被拉的蛋白）都没曝出来，请各位高手知道一下吧，谢谢。本人western 没有问题。
如果连对照都没有出来，就首先应该考虑程序流程的问题，无论是否发生了pull down，起码对照应该成立不是么？然后再考虑的是相互作用的蛋白的问题。
if the input is not good, there are several reasons:
1.for the western procedure, check if primary antibodies, secondary antibodies are good, make all buffer and solution freshly and try
2.for the pull down assay, what kind of beads you are using, maybe there isn't good binding for your beads and target protein, if you are using protein A/G agrose, check if the pull down antibodies is ok.
3. make sure the cell lysate you use has enough total protein and relatively high expression of your target protein
etc.
1.for the western procedure, check if primary antibodies, secondary antibodies are good, make all buffer and solution freshly and try
2.for the pull down assay, what kind of beads you are using, maybe there isn't good binding for your beads and target protein, if you are using protein A/G agrose, check if the pull down antibodies is ok.
3. make sure the cell lysate you use has enough total protein and relatively high expression of your target protein
etc.
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