某一种蛋白在总蛋白中含量很低做western很难做得出来除了加大上样量外还有哪些可以让成功的几率提高呢

Question

某一种蛋白在总蛋白中含量很低 做western 很难做得出来 除了加大上样量外 还有哪些可以让成功的几率提高呢

Answer 1

Use more concentrated antibody.Incubate longer (e.g.overnight) at 4ºC.
Use an enrichment step to maximize the signal (e.g.prepare nuclear lysates for a nuclear protein,etc.).
Do not over wash the membrane.
Too much blocking does not allow you to visualize your protein of interest.
Instead of using 5% milk in the antibody buffers try removing the milk or using 0.5%.Switch blocking reagents or block for less time.