In initial studies that utilized Y2H, proper controls for false positives (e.g. when DB-X activates the reporter gene without the presence of AD-Y) were frequently not done, leading to a higher than normal false positive rate. An empirical framework must be implemented to control for these false positives. Limitations in lower coverage of membrane proteins have been overcoming by the emergence of yeast two-hybrid variants, such as the membrane yeast two-hybrid (MYTH) and the split-ubiquitin system, which are not limited to interactions that occur in the nucleus; and, the bacterial two-hybrid system, performed in bacteria;