Methodology for Cell Fractionation

Isolation of chloroplasts

Isolated chloroplasts are required to study the electron transport system of the photosynthetic apparatus. There are as many techniques as there are research groups in the field of chloroplast research. Any one of these methods can be followed, provided the isolated chloroplasts are biochemically active as the chloroplasts in vivo. A general procedure is given below.





Principle

The cell organelles, depending upon their size and weight, sediment at different centrifugal fields.


Materials
» Isolation Medium
Weigh 2.42g Tris (20mM); 72.8g sorbitol (0.4M); 1.168g NaCl (20mM); 0.610g MgCl2 6H2O (3mM) and dissolve in one liter of distilled water. Adjust to pH 7.8.


Procedure
1.
Cut 5 to 10g of leaf tissues into small bits. Add 20mL of the prechilled isolation medium.
2.
Homogenize with an omnimixer (three five-seconds with five seconds intervals).
3.
Filter the brei through eight-layered cheese cloth.
4.
Centrifuge at 3000g for 2 min.
5.
Discard the supernatant and suspend the pellets in the isolation medium.
6.
Centrifuge at 3000g for 2 min.
7.
Discard the supernatant and resuspend the pellet in a small volume of the grinding medium and store on ice.
8.
Since any further advanced study on isolated chloroplasts is expressed on the basis of chlorophyll, estimate the chlorophyll by diluting 0.1-0.2mL of chloroplast suspension to a total volume of 4mL with 80% acetone.
Calculate the chlorophyll content as follows:
(12.7 x A663)             -               (2.69 x A645)           = chl.a (mg/mL)
(22.9 x A645)             -               (4.68 x A663)           = chl.b (mg/mL)
(20.2 x A645)+              (8.02 x A663)= total chl. (mg/mL)
Calculate  the chlorophyll  concentration of the  stock chloroplast suspension.





Notes

1. An alternative grinding medium, most useful for many purposes is
330mM sorbitol
10mM Na4P2O7 (Sodium Pyrophosphate)
5mM MgCl2  2mM Na isoascorbate
Adjust to pH 6.5 with HCl. Centrifuge at 6,000rpm.
2. Most leaves yield better chloroplasts if freshly harvested except spinach, which can be stored for four weeks in cold for better yield. If leaves are brightly illuminated for 20-30 min prior to grinding, the chloroplast yield is increased.




References

1. Procedure Manual Plant Sciences Division School of Biological Sciences M K University Madurai (1982).
2. Walker, D A (1980) In: Methods in Enzymology 69 (Eds Colowick, S P and Kaplan, N O) Academic Press p 94.