The GUS assay does not require the presence of any cofactors or ions for function. Beta-glucuronidase can function through a wide range of pH values, and is fairly resistant to thermal inactivation. However, GUS is susceptible to inhibition from certain heavy metal ions, such as Cu and Zn.
Additionally, the interpretation of the assay is limited by the movement of diX-indigo throughout the cell. DiX-indigo, can associate with lipids to diffuse far from the site of enzyme activity, which shows a lack of cytosolic localization and irregularity of substrate penetration. This can potentially lead to an incorrect interpretation of GUS protein localization. Despite a lack of cellular localization, nuclear localization of GUS has been well observed. GUS assays can be carried out in the presence of potassium ferricyanide to prevent the stain from diffusing.