小中大
我最近用非变性裂解液又做了一次，HSP90大部分都沉淀下来了，但是杂AKT，看到的结合量还是很少，请大家帮我想想还有什么地方有可能破坏了蛋白之间的相互结合？比如清洗珠子的时候能不能不用裂解液，用PBS可以吗？我觉得这样比较温和。谢谢了！
1. You can decrease the concentration of salts and detergent, e.g. 50mM Tris HCl pH 7.5, 150 mM NaCl, 0. 1% Triton X-100/NP-40, and using 1/3-1/5 of proteinase/phosphase inhibitors after first washing because most non-binding proteins are washing away.
2. You can use linker reagent to treat sample if you are so worry about decrease of PPI.
另外，我还想问一下：coIP阴性对照是怎么设的？我们实验室没有正常的IgG,应该用什么设阴性对照呢？是不是应该设一个只加珠子不加抗体的对照啊？来证明珠子不会将蛋白沉下来。
Typical controls for co-IP are
1. Normal Ab isotype as Ab used in IP.
2. Use same cell/tissue from knock out animal.
3. 只加珠子不加抗体 only for pre-cleaning cell/tissue lysates, it can not be used as control.
4. 我们实验室没有正常的IgG: You can ask your supervisor to buy it, it is cheap.
