RoC

That's great. Thanks for that. Much appreciated. RoC

Hi there, I would like to carry out an enzyme activity assay for the galactosyltransferase enzyme in the Golgi of CHO cells. I found a paper with a protocol using tritium labelled UDP-Glucose as donor substrate. I've tried to contact the authors for more details but have gotten no reply. As its my first time working with radioactivity I would like to know if my dilution calculations of radiolabeled glucose are ok or are totally incorrect? Your advice is greatly appreciated. The protocol suggests using 0.5 mM Glucose at 50,000 dpm / nm in a 25 ul assay. I take it the dilution must first by made up for the radiolabeled glucose with non-labeled glucose before addition to the final assay volume. I was thinking of making up a 1ml volume stock at 5 mm UDP-Glucose at 50,000 dpm /nm. Calculations for UDP-[3H]Glucose in GalTI assay: Radioactive Concentration (RAC): 1000 µCi/ml Specific Activity (SA): 15 Ci/mmol Volume of final Assay mix: 25 µl Desired concentration of final diluted mix: 0.5 mM Specific Activity: Equation to convert Ci/mmol to DPM/fmol: DPM/fmol = [specific activity (Ci/mmol) x [2.22 x 1012 DPM/Ci] x [mmol/1012 fmol] = SA x 2.22 SA = 15 Ci/mmol DPM/fmol = SA x 2.22 = 15 x 2.22 = 33.3 DPM/fmol = 33.3 x 106 = DPM/nmol = 33,300,000 DPM/nmol Protocol suggested to use UDP-[3H]Glucose at 50,000 DPM/nmol Nominal Stock Concentration [Radioligand] = RAC/SA = (1000 µCi/ml / 15 Ci/mmol) 1 Ci/106 µCi = 66.6 x 10-6 mmol/ml = 66.6 µmol = 66.6 µM 66.6 µM = 66600 nmol / L therefore 1ml of stock = 66.6 nmol 1ul = 0.666 nmol And if 33.3 x 106 dpm/nmol (from specific activity) then there is 2.2 x 107 dpm / ul of hot stock Dilution What volume (ul’s) of radioactive UDP-Glucose is required to be added a 1 ml solution of 5 mM UDP-Glucose to get a concentration of 5 mM UDP-Glucose at 50,000 dpm / nm? 5mM stock concentration in 1 mL volume for addition to assay 5mM / 1ml = 5 x 103 nmol / ml 5,000 nmol / ml @ 50,000 dpm / nm = 2.5 x 108 dpm 2.5 x 108 ÷ 2.2 x 107 = 11.3 ul of hot stock to be added to 1 ml of 5 mm cold UDP-GlcNAc to get a concentration of 50,000 dpm / nm.