Personally, I suggest that the time should not be longer, and then the protein may be denatured causing the subsequent experiments to be impossible. I originally encountered this kind of problem is to use the following methods to try to solve, you can also try (1) check how your sandwich structure sandwich, especially the membrane and filter paper edges are not short joints, this is the most likely to occur problems. In addition, there is no air bubble between the membrane and the adhesive, it is not recommended to roll the membrane and the adhesive with external force, it is recommended to wet the membrane and the adhesive with buffer and then drive out the gas from one side of the cover under the cover, just like making a coverslip for a slice. Be absolutely sure that there is no shorting and gas bubbles, otherwise it is easy to have the problem of incomplete membrane transfer. (2) Switch to a constant pressure try, this program I do not know what the principle is, but sometimes it does solve the problem. (3) Switch to the semi-dry transfer method, but note that the semi-dry transfer should ensure that there is no shortening of air bubbles, and the time can not be too long, semi-dry transfer of a lot of heat. I'm not sure if your protein needs to be active, if it is inactivated by heat, then you can't use this method. (4) This method is more wasteful, depending on the boss to let you use. Change the finished buffer product, Sigma's is better. In addition to change the 24-layer filter paper finished products, use once do not use a second time. There is also also a brand of membrane, our laboratory once encountered a brand of membrane completely turned out of the phenomenon. Above, I hope your problem can be solved soon.