Alienation in Of Mice and Men is everywhere. As soon as Lennie and George get to the ranch, Slim remarks that he has not seen too many people travel together. This makes George and Lennie's friendship an anomaly. Here is what Slim says:
Slim looked through George and beyond him. “Ain’t many guys travel around together,” he mused. “I don’t know why. Maybe ever’body in the whole damn world is scared of each other.”
Slim's point is that alienation is the norm in this world.
We also see alienation on the ranch in the person of Crooks. When Lennie comes into Crooks' living quarters, a conversation ensues. In this conversation Crooks says that no one have ever visited his dwelling. In short, Crooks lives a life apart from the other men. Moreover, Crooks is not allowed to enter into bunkhouse of the other men to spend time with them. So, we can say that Crooks is completely alone.
Finally, we can also say that this alienation extends to every other person on the ranch. There simply are no friendships, not even in marriage (there is complete tension between Curley and his wife).