Figure 1.

Schematic Overview of the Basic Principles of HEK293 Amenability Assays

HEK assays can be separated into five steps. Step 1: cloning of GLA mutations using site-directed mutagenesis. Step 2: transfection of seeded HEK293 cells with a mock control (negative control; necessary for AGAL background substraction), wild-type AGAL (positive control; reference activity is set to 100%), and the GLA mutation of interest. Step 3: incubation of cells with or without DGJ. Step 4: cell lysis to release overexpressed AGAL. Step 5: AGAL activities are measured and compared to each other. Depending on the assay protocols, various sub-steps can be included in between, such as wash-outs, determination of transfection efficiencies, among others. AGAL/GLA, α-galactosidase A; DGJ, 1-deoxygalactonojirimycin; WT, wild-type.