Most current high throughput puriWcation procedures for the green Xuorescent protein (GFP) suVer from poor yields and low purity. An improved puriWcation procedure that delivers highly pure protein (>95% homogeneity) in high yields (>70% of the initial Xuorescent protein content) has been developed. The puriWcation procedure requires only two steps: the cell lysate is heated to 60 Í for 4min in ammonium sulfate and triethylamine, followed by hydrophobic interaction chromatography using isopropanol during the elution phase. The resulting pure product exhibits the same Xuorescence proWle as the crude sample. This procedure has been demonstrated on three commercial variants of GFP from Aequorea victoria, enhanced green, enhanced yellow, and enhanced cyan Xuorescent protein (Becton-Dickinson). The yield and purity of material are superior to other recently described methods.