nate Dehydrogenase Activity SDH Assay 1 Allow the
Last updated: 9/19/2023
nate Dehydrogenase Activity SDH Assay 1 Allow the spectrophotometer to warm up for at least 5 min The wavelength should be set at 600 nm 2 You will be assaying your fractions F S1 P1 S2 and P2 for mitochondrial activity Then you will assay P2 in addition to three controls Malonate azide and succinate controls using a P2 Blank Assay the fractions according to the table below Table 1 All fractions should be kept on ice 3 Gather cuvettes labeling each using the table below To all cuvettes add the various solutions listed across the top of table 1 except for the subcellular fraction sample First add the correct volume of assay buffer to all cuvettes Then in the same manner add the volumes of azide DCIP malonate and succinate indicated in the table on the following page Cover each cuvette with Parafilm and gently invert twice to mix the contents 4 After mixing together the contents of the first five columns thoroughly homogenize the subcellular fraction sample with a Pasteur pipette 5 Add the appropriate volume of the subcellular fraction sample to all of the BLANKS ONLY cover the cuvettes with parafilm and invert twice to mix the contents 6 TIMING IS CRITICAL for this step For each fraction sample you will be measuring the Abs at 600 nm at time zero and at 3 minutes For each sample you will blank the spectrophotometer and then add your subcellular fraction to the sample cuvette invert to mix and then immediately measure the Abs at 600 nm To ensure that all reactions proceed for exactly 3 min start your timer counting up immediately after adding the subcellular fraction to the sample cuvette When the timer reaches 3 min for the sample measure the Abs at 600 nm blanking first Do not invert the cuvette to mix prior to the 3 min Abs measurement Proceed to measure all remaining samples in the same way recording data in your lab notebook 7 Later you will create a bar graph of the velocity of SDH in each fraction and the controls e the handout provided by your TA to calculate the velocity or rate at which DCIP is being uced by SDH These data will be used to make a bar graph of your velocities