Chloroplast Isolation
by Differential Centrifugation
Week 12th
Kang Ji-Seon
Dept. of Biochemistry
Genome Regulation Center
Yonsei Univ.
Contents
Centrifugation
• Differential centrifugation
• Density gradient centrifugation
- Isopycnic centrifugation
- Rate zonal centrifugation
Types of centrifugation
• Low-speed centrifuge
• High-speed centrifuge
• Ultracentrifuge
Centrifuge Rotor
• Swing-out rotor
• Fixed-angle rotor
• Vertical rotor
< Background info.>
Chloroplast Isolation
• Protocol
- Chloroplast isolation
- Estimation of chlorophyll conc.
- Estimation of the percent of
intact chloroplasts
Further study
< Experiment.>
Centrifugation
Centrifugation is a process that involves the use of the centrifugal force for theseparation of mixtures used in laboratory, increasing the effective gravitationalforce on a test tube so as to more rapidly and completely cause the precipitate(pellet) to gather on the bottom of the tube. The solution (supernatant) is theneither decanted or used in other step.
The rate of centrifugation is specified by the acceleration applied to the sample,typically measured in RPM or g.
There are various types of centrifugation
• Differential centrifugation
• Isopycnic centrifugation
• Density gradient centrifugation
Differential centrifugation
Differential centrifugation is a procedure in which the homogenate is subjectedto repeated centrifugations each time increasing the centrifugal force.
Isopycnic centrifugation
Isopycnic centrifugation is a technique usedto separate molecules on the basis of density.(The word "isopycnic" means "equal density".)
Typically, a "self-generating" density gradientis established via equilibrium sedimentation,and then analyte molecules concentrate asbands where the molecule density matchesthat of the surrounding solution.
* Text book : example of DNA separation
CsCl2 is used because at a conc. of 1.6 to1.8g/mol is very similar to the density of DNA.
Rate zonal centrifugation
(e.g. sucrose gradient centrifugation)
Density of the particles being separated
are greater than the density of the solvent.
Separation is based upon mass (i.e., larger
particles will sediment faster).
Typically, a sucrose density gradient is
created by gently overlaying lower conc. of
sucrose on higher conc. in a centrifuge tube.
During centrifugation, the particles travel
through the gradient until they reach the
point in the gradient at which their density matches that of the surrounding sucrose.
This fraction can then be removed and subjected to further analysis.
Types of centrifugation
• Low-speed centrifuge
- table centrifuge
- typical maximum speed is 6000rpm.
- room temp.
- cell, nucleus, etc. (easily precipitated material)
• High-speed centrifuge
- max. speed 20000-25000rpm (60000 X g)
- temp. control
- cell, nucleus, organelle, etc.
• Ultracentrifuge
- max. speed 9800km/s2 (1000000 X g)
- temp. control and vacuum system
- cell, nucleus, organelle, components of memb., polysome, macromolecule, etc.
Types of Rotor
Swing-out rotor
Fixed-angle rotor
Vertical rotor
Ferricyanide Photoreduction
- Measure of intact chloroplasts
This assay is based upon the inability of the ferricyanide to cross thechloroplast envelope and to react with the electron transport system in thethylakoid membs.
Ferricyanide reduction, as indicated by the decrease in the absorbancy at410nm, occurs only when ruptured chloroplasts are present in the preparation.
The degree of integrity of the chloroplast preparation is assessed by comparing
the rate of ferricyanide reduction upon illumination before and after osmoticshock of the chloroplasts.
Analysis of the results, presented here, indicates that 88% of the chloroplasts
in the spinach chloroplast preparation are intact.
Chloroplasts (equivalent to 25g/ml chlorophyll) were illuminated in the
presence of 1.5mM ferricyanide. The reduction of ferricyanide was measured
spectrophotometrically (410nm).
Graph A demonstrates the change in absorbancy of the two samples (before and
After osmotic shock) during 6 minutes.
Graph B shows bars representing the slopes of the lines in Graph A.
Procedure - 1. chloroplast isolation
• Perform all steps as 2 to 4℃
• Use pre-cooled buffers and equipment. All centrifugations should be performed
at 2 to 4℃ with pre-cooled rotors.
1. Wash 5g of leaves thoroughly with DW and remove the excess water.
Remove the midrib veins and cut the leaves into small (1cm) pieces.
2. Place the leaf pieces into a pre-chilled mortar.
Add 22ml of 1X CIB buffer with BSA (4ml/g of leaves).
3. Grind them until the leaf pieces change to slurry.
4. Squeeze the leaf slurry through 8 layers of gauze into a pre-chilled 50ml tube.
Decant 1ml of homogenate into 1.5ml tube.
5. To remove unwanted whole cells and cell wall debris, centrifuge the tubes for
3mins at 200 X g. A white pellet (P1) is precipitated.
6. Transfer the supernatant to fresh, chilled 50ml tubes and centrifuge for 7 mins
1000 X g to sediment the chloroplasts as a green pellet (P2). Don’t forget to
leave 1ml of S1 before centrifugation.
7. While centrifugation, resuspend the P1 pellet in 1ml of 1X CIB with BSA.
8. Decant the supernatant this time into new tube as a S2 and gently break the
pellet (P2) by finger tapping. Resuspend the P2 of tube in 1ml of 1X CIB with
BSA by gently pipetting up and down. Avoid foaming.
Decant 20ul of the resuspended pellet (P2) into 1.5ml tube.
9. Purification of intact chloroplast. At this step the intact chloroplasts can be
separated from the broken chloroplasts by centrifugation on top of a
40% of Percoll layer.
a. Carefully overlay the chloroplast suspension (P2) on top of the 10ml 40%
Percoll and centrifuge for 6mins at 1700 X g. The broken chloroplasts
will form a band on top of the Percoll layer and the intact chloroplasts
will sediment to the bottom as a small green pellet.
b. Carefully decant the supernatant (S3) into new tube as a S3 and keep
the pellet.
c. Resuspend the pellet (P3) in 0.5ml of 1X CIB without BSA as a P3.
(The chloroplast suspension should be kept in the dark, on ice, until
further use.)
Procedure – 2. Estimation of chlorophyll concentration
1. Add 10ul of each sample (homogenate, S1, P1, S2, P2, S3 and P3) to 1ml of
an 80% acetone solution and mix well.
2. Centrifuge for 2mins at 3000 X g. Retain the supernatant.
3. Measure the absorbance of the supernatant at 652nm. Use the 80% acetone
solution as the reference blank.
4. Multiply the absorbance by the dilution factor (100) and divide by the
extinction coefficient of 36 to obtain the mg of chlorophyll per ml of the
chloroplast suspension.
Procedure – 3. Estimation of the percent of intact chloroplasts
:: Ferricyanide photoreduction
A. Without osmotic shock
: Mix 100ul chloroplasts (P3) with 900ul of 1X CIB.
Add 15ul of 100mM ferricyanide*(final conc. 1.5mM).
B. With osmotic shock
: Mix 100ul chloroplasts (P3) 450ul of water.
Incubate for at least 15 seconds to allow for osmotic shock.
Add 450ul of 2X CIB and 15ul of 100mM ferricyanide* (final conc. 1.5mM).
* Prepared freshly in DW.
1. Place the tubes in a glass beaker filled with ice water.
2. Illuminate with a closely positioned 40W bulb. Take a 1ml sample before
illumination, and then one every 2 mins after illumination.
Measure the absorbance at 410nm using a spectrophotometer.
Continue illumination for 6 mins.
3. Photoreduction of ferricyanide results in a decrease of the absorbance at
410nm. Plot the absorbance at 410nm versus the time. The rate of
decrease in absorbance of each sample is the slope (A410/minute) for the
graph. Calculate the slope for each reaction (A and B).
4. To calculate the percent of intact chloroplasts, use the following formula:
Further study
1.Explain about the rpm , rcf and g-force and describe their differences.
2.Why spinach (plant) should be kept in the dark to get a optimal yield of
intact chloroplast?
3. What is a role of BSA (Bovine Serum Albumin) in CIB? -> Bonus Q!!
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