Subcellular fractionation is a biotechnological procedure

Last updated: 9/19/2023

Subcellular fractionation is a biotechnological procedure that separates subcellular components by a series of increasing centrifugation speeds The procedure begins by centrifuging cell lysate broken cells at a low centrifugation speed At lower centrifugation speeds only the densest cellular components will sediment form a pellet while lighter components remain in the supernatant non pellet soluble phase The supernatant can then be separated from the pellet and centrifuged at a higher speed to sediment the lighter cellular components still in solution This incremental increase in centrifugation speeds is known as differential centrifugation Differential centrifugation is a means by which subcellular components can be separated on the basis of their different densities Thus subcellular fractionation uses differential centrifugation to separate different subcellular components on the basis of their varying densities In this lab we will seek to separate nuclei from mitochondria found in plant tissues Since nuclei are more dense than mitochondria we will be able to separate these structures using centrifugation on the basis of their differing densities Centrifugation at a lower speed will sediment the dense nuclei which will form the pellet while the less dense mitochondria remains in the supernatant Centrifuging the filtrate filtered cell lysate separates the filtrate into two fractions supernatant and pellet Each fraction is then tested for the presence of nuclei and mitochondria The presence of nuclei will be assessed via microscopy whereas the presence of mitochondria will be tested for by assaying for the SDH succinate dehydrogenase enzyme which is an enzyme that is specific to mitochondria Subcellular Fractionation of Cauliflower is a 1 week long experiment First you will grind cauliflower to break open cells and then perform differential centrifugation of the cauliflower tissue You will then complete microscopy on various fractions and perform an SDH assay and analyze the results