Isolation of Chloroplast from Spinach

To prepare the cell tissues for fractionation, they were first minced and suspended in an isotonic solution. All containers and solutions were made ice-cold to maintain the integrity of enzymes and organelles.  Due to the different densities and sizes, cells have different centrifugal forces. With the rpm and distance (x), it was possible to determine the RCF.

RCF=1.119*10-5 (RPM)2x

Differential Centrifugation

            When carrying out the differential centrifugation, the homogenate was centrifugated several times at higher speeds sedimenting it into smaller particles. The first centrifugation was carried out for 600g at 10 minutes. The next procedure was carried out for 10, 000g at 30 minutes with a higher speed centrifuge. In the case of spinach cells, the filtrate is centrifuged at 200g for 1 minute. To sediment the chloroplasts, the supernatant is span with the same procedure at 1300g for 5 minutes.

Density Gradient Centrifugation

            For a better resolution of cells and organelles, the Density Gradient Centrifugation method was applied. The homogenate was added in a centrifuge tube with a highly concentrated sucrose solution. As the tube spin, the organelles distributed themselves in the solution depending on the size of the organelles. With a centrifugation of 10, 000g at 20 minutes, it was easy to identify nucleic mitochondria layers.

Determining centrifugal speeds and isolation of chloroplast

            The rotor radius was measured from the bottom and the centrifuge tube is placed horizontally in the bucket rotor. The rpm was then calculated and the centrifugations were used to deduce chloroplast fractions. The centrifuge tachometer was then used to determine the centrifuge speed for each experiment.

            After removing the major veins of spinach, 4g of the remains were weighed, cut into small pieces and ground for 2 minutes after mixing it with 15ml of a cold Tris-NaCl buffer. The suspension was then filtered in a suspension tube and centrifuged at 200g for I minute. The centrifuge was then decanted and span at 1300g for 5 minutes to ensure balancing. Finally, the centrifuge tube was covered and inserted in an ice-water bath.