User:Carly M. Montanero/Notebook/CHEM-571/2013/10/09

Biomaterials Design Lab: Fall 2013

Main project page Previous entry      Next entry

Objective To observe and measure ADA turnover kinetics in the presence of an inhibitor, EHNA. This work will be the basis of our comparison to ADA-AuNP turnover studies. From Dr. Hartings. Procedure Creating the Adenosine Stock Add 0.1073 g of adenosine to a 10 ml volumetric flask. Fill with 50 mM phosphate buffer at pH 7.4. This created a stock solution of 0.04 M. Add 10.00 μL of the 0.04 M adenosine stock to a 10 mL volumetric flask. Fill with 50 mM phosphate buffer at pH 7.4. This created a final solution of 40 μM. Enzyme Kinetics Measurement Add 3 mL of 40 μM adenosine solution to the cuvette and 30 μL of inosine. Start kinetics measurement: 1 ms integration 10 scan average Set "Save the first available scan every" to 15 seconds Set "Stop after this amount of time" to 10 minutes Set "File Type" to Tab Delimited Just before 1 minute, add 1.5 μL of 1 nM EHNA. Figures Notes EHNA stock 5mg EHNA in 1mL DMSO ---> 15.9mM EHNA (1.9μL)(15.9mM EHNA)=(10mL)(C2). C2 ---> 3μM EHNA The reaction samples will contain roughly 1nM EHNA.