Isolation of Chloroplasts from Spinach Leaves by fractionation

Theory

  

Chloroplasts are organelles 5-10 um in size in plants which perform the function of photosynthesis.  These structures are mainly located in the palisade parenchyma of the mesophyll cells in the leaf. Chloroplasts belong to a family of specialized organelle called the plastid. The stem, leaves and unripened fruit in all the plants contain chloroplast. But leaves are major part of plants performing the process of photosynthesis. The green color of these structures in plants is due to the presence of a pigment called chlorophyll which resides in the chloroplasts. 

Chloroplasts are found in the mesophyll cells of the leaves. The chloroplast is divided into three compartments bounded by three membrane systems: an intermembrane space between the inner and outer membranes, the stroma and the thylakoid lumen.  Chloroplasts have a double membrane structure called the chloroplast envelop.  The chloroplast envelop has an inner membrane and an outer membrane. A third membrane system called the thylakoid membrane surrounds the thylakoids in the stroma.  

Chloroplasts are located inside the thylakoid membranes.  Thylakoid membrane consists of the thylakoids which are flattened discs arranged on top of the other and they are termed as grana.  The thylakoids are located inside the stroma. Photosynthesis takes place in the thylakoid membranes. The chlorophyll molecules absorb light in the form of photons and this leads to the emission of electrons by the chlorophyll molecules. This drives the hydrogen ions across the membrane surrounding the thylakoid stack. This leads to the formation of an electrochemical gradient which drives the production of ATP. 

Principle

The cells of spinach leaves are disrupted mechanically by using a blender or homogenizer, freeing the untethered organelles, which can then be sorted out from each other by filtration and differential centrifugation as cell fractions. Filtration will remove large debris (e.g., cell walls) and unbroken cells, providing a filtrate that contains organelles (nuclei, chloroplasts, mitochondria, ribosomes), small membrane vesicles, and soluble components; most of these will not be visible in the light microscope. Low-speed centrifugation will sediment remaining large bodies from the filtrate, and moderate-speed centrifugation will sediment chloroplasts, leaving mitochondria and ribosomes and soluble components in the supernatant. (The mitochondrial fraction could be collected by high-speed, and ribosomal and membrane vesicle fractions by ultra-high-speed centrifugation.) Repeated rounds of differential centrifugation can be used to further purify the chloroplasts when highly purified preparations are required.

Procedure:

Keep the leaf and all fractions ice cold throughout steps 1 - 6 and 10.  

1. Take 8 grams of de-veined leaf tissue rinsed in ice water, blotted and cut into pieces about 1 cm square.
2.  Place the leaf pieces in a pre-chilled blender cup containing 40 ml of ice-cold 0.5 M sucrose. 
3. Blend for 15 sec. at top speed, pause about 10 sec., then blend again for 10 sec. 3.
4. Remove the ice from the 100-ml beaker, then squeeze the leaf homogenate through four layers of pre-chilled cheesecloth into the cold beaker by twisting the top corners of the cloth around each other.
5. Pour 14 ml of the homogenate into each of two centrifuge tubes and centrifuge at 200g for 5 min.
6. Using a Pasteur pipet, transfer each supernatant (containing the chloroplasts) to a second centrifuge tube and centrifuge at 1000g for 7 min.
7.  Using the pipet, discard the supernatant but be careful not to disturb the pellet.
8. Pour 2 ml of phosphate buffer onto the pellet and gently suspend it by moving it up and down in the pipe.
9. Using a clean Pasteur pipet, add buffer until you have a total volume of 8 ml and mix the diluted suspension using the pipet. This is the chloroplast suspension.
10. Examine under the microscope.
11. Using a hemacytometer, determine the number of chloroplasts per mL of suspension media or observe mitochondria under microscope.

Estimation of chlorophyll a concentration of the suspension.

1. Take 4.75 ml of 80% acetone in tube tube.
2. Add 0.25 ml of chloroplast suspension, mix well, and read the Centrifuge at 3000xg for 2 minutes.
3. Take 1000ul of the supernatant and transfer into a cuvette and measure the absorbance at 650 nm. Use 80% acetone as blank.
4. Take the average of the two values and estimate the mg/ml chlorophyll concentration using the following formula:

A at 650 x df /36 = mg/ml chlorophyll.  

Where A 650 is the absorbance at 650 nm and 36 is the extinction coefficient of chlorophyll.

References:

Joly D and Carpentier R (2011). Rapid isolation of intact chloroplasts from spinach leaves. Methods Mol Biol. 684:321-325.

Tetsuko Takabe, Mikio Nishimtjra and Takashi Akazawa (1979). Isolation of Intact Chloroplasts from Spinach Leaf by Centrifugation in Gradients of the Modified Silica “Percoll”. Agricultural and Biological Chemistry, 43(10): 2137-2142.

Extraction of Plasmid DNA from bacteria (Alkaline lysis method)

Principle:

The bacterial cells are lysed with lysozyme and SDS at high pH and the lysate is then neutralized. The plasmid undergoes renaturation but not the chromosomal DNA. The later gets precipitated out in the form of protein-DNA-SDS complex. Subsequent deproteinization with phenol: chloroform, plasmid DNA is precipitated with ethanol by spinning at high speed. 

Requirements:

1. Overnight Log- phase culture in LB broth (10 g tryptone, 5 g yeast extract and 10 g NaCl, 1L DW)
2. Solution I (Lysis buffer): 25 mM Tris, 50 mM Glucose, Lysozyme (GNB: 0.5 mg/ml, GPB: 3-5 mg/ml), pH 8.0
3. Solution II (Lysis): 0.2M NaOH, 1% SDS 
4. Solution III: 3 M Sodium acetate, pH 4.8 by acetic acid
5. Phenol: Chloroform (1:1)
6. RNase A: (1 mg/ml in 5 mM Tris-HC1, pH 8.0)
7. Absolute ethanol/Isopropanol
8. 70% ethanol
9. TE buffer: 10 mM Tris, 1 mM EDTA, pH 8.0; Autoclave before use
10. Water bath
11. Cooling centrifuge (upto 20000 rpm)
12. Micropipettes (1 – 10, 10 -100, 100 – 1000 µl ) and sterile tips

 Procedure: 

1. Take 1.5 ml of overnight LB-broth culture of bacteria in a MFT and spin at 5000 rpm for 2 min.
2. Remove the supernatant and spin once with same volume as above to collect more cell mass.
3. Remove the supernatant.
4. Add 100 µl Sol. I. Re-suspend and keep for 30 min at RT.
5. Add 200 µl freshly prepared Sol. II and mix gently (Do not vortex). Keep for 5 min at 4°C.
6. Add 150 µl ice cold Sol. III and mix gently. Keep in ice bath for 10 min.
7. Spin at 10000 rpm for 5 min at 4°C. Collect Sup-T into new MFT.
8. Add 1-2 µl of RNase and incubate for 10 min at RT.
9. Add equal vol. of Phenol:Chloroform (1:1), mix gently and keep for 10 min at RT.
10. Centrifuge at 8000 rpm for 10 min at 4ºC and collect the supernatant in a new sterile MFT.
11. Add two vol. of absolute ethanol and stand it for 10 min in cold.
12. Spin at 13000 rpm at 4ºC for 10 min.
13. Remove the Sup-T into new MFT and add 1000 µl of 70% ethanol. Mix well and keep at 4°C for 30 min.
14. Spin at 13000 rpm at 4ºC for 10 min. Remove Sup-T.
15. Dissolve the pellet collected during spin in 50 µl TE and store in deep freeze.