被问到一个问题,不知道怎么回答,特来版上求助。 Immunostaining showed two proteins co-localize in the membrane, question is to confirm that the two proteins directly bind each other. First, I was thinking about co-ip, but this method does not exclude the possibility other proteins are also involved, so it's hard to tell direct protein binding with co-ip. Second method I was thinking about is yeast two-hybrid, which is just to detect the interaction of two proteins. But drawback is false positive. Third method is a combination with co-ip, mass spectrometry. co-ip will show both proteins are in one complex, and ideally if no other proteins are involved, mass spectrometry will only show the two proteins that are studied. The fourth method is BiFC, label both proteins with half of GFP, when they bind together, there will be GFP signal. Problem for this method is that can we say the two proteins directly bind with each other? 请教大家的建议,谢谢!