In lab, our objective was to obtain a mitochondrial enriched
extract from cauliflower, use the mitochondrial enzyme succinate
dehydrogenase as a marker for mitochondrial enrichment, and provide
a better understanding of the activity of enzymes using succinate
dehydrogenase as an example. Part 1 was: Enrichment of mitochondria
from cauliflower extract by differential sedimentation and Part 2
was: Monitoring of fractions for specific activity of the
mitochondrial enzyme succinate dehydrogenase. What is the general
differential centrifugation scheme you perform in the differential
centrifugation and SDH Activity lab? Why are the fractions isolated
not considered pure but instead defined as “enriched”? A scientist
recently identified a nuclear protein involved in nuclear lamina
assembly. Which fraction of your differential centrifugation scheme
would be the most enriched fraction for your protein of interest?
Why? Why do you think it is important to keep the cell fractions on
ice? How did you detect the enrichment of mitochondria in the
isolated fractions? Which of the fractions you collected should
contain the highest enrichment of mitochondria? Does your specific
activity of your fractions support this? In order to calculate the
specific activity of the isolated fractions, the protein
concentration has to be determined. Why is the protein
concentration necessary for accurate comparison of the enzyme
activity of the fractions?