HSD17B4 biochemical assay optimization. (A) Results from timecourse experiment for biochemical meaures of HSD17B4 activity. Graph shows fmol E1/μg protein produced after brains were incubated for 10 minutes, 30 minutes or 90 minutes with and without cold trap. (B) Results from dose response curve for biochemical meaures of HSD17B4 activity. Graph shows fmol E1/μg protein produced after incubation of brain with 10 nM, 50 nM, 100 nM, 300 nM of 3H-E2 substrate for 5 minutes. Each point represents the average of duplicate measurements for each substrate concentration. 50 nM 3H-E2 substrate is centered in the linear rise of enzyme activity.