Citrate Synthase Activity Assay: A Colorimetric Assay to Measure Mitochondrial Citrate Synthase Activity in Drosophila Tissue Homogenate
Transkript
Transfer 10 adult fly thoraxes to 100 microliters of ice-cold extraction buffer immediately, and homogenize them with a pestle on ice. Keep the samples cold by homogenizing for five to 10 seconds on ice, letting them sit on ice for five seconds, then, repeating until all tissues are completely homogenized.
Aliquot 10 microliters of each homogenized sample into a new tube for protein content measurement, keeping these tubes on ice as well. Add 400 microliters of ice-cold extraction buffer to each remaining sample for a total volume of 500 microliters, and pipette up and down gently to mix.
For each reaction, add 1 microliter of diluted cell lysate to 150 microliters of the freshly prepared reaction solution. Mix thoroughly by gently pipetting, making sure to avoid bubble formation. Then, use a plate reader to measure the absorbance at 412 nanometers every 10 to 30 seconds for four minutes at 25 degrees Celsius.
Plot the data as absorbance over time, then, calculate the slope for the linear portion of the curve. Finally, divide the reaction rate or slope by the sample protein concentration to normalize the citrate synthase activity.
