Isolation and preparation of plant chloroplasts
The isolation and preparation of plant chloroplasts is an important technical condition for the study of the structure and function of chloroplasts and the mechanism of photosynthesis with active isolated chloroplasts. This experiment is to learn the technical method of isolation and preparation of chloroplasts.
Principle
The basic principle of the separation and preparation of plant chloroplasts is due to the fact that chloroplasts have a relatively fixed size, shape and density, which determines their particular sinking rate during centrifugation in the division. Taking advantage of the fact that chloroplasts have a different diameter and sedimentation coefficient than other organelles, they are separated by centrifugation in a graded manner. The homogenate obtained by grinding the leaves is filtered and centrifuged to prepare chloroplasts. The periplasm of chloroplasts is relatively fragile, and the isolation of chloroplasts should be carried out in isotonic buffer solution at a temperature of 0~4 ℃. The viability of chloroplasts decreases as the time of isolation increases, therefore, the isolation should be completed in as short a time as possible.
Operation method
Isolation and preparation of plant chloroplasts
Principle
The basic principle of the separation and preparation of plant chloroplasts is due to the fact that chloroplasts have a relatively fixed size, shape and density, which determines their particular sinking rate during centrifugation in the division. Taking advantage of the fact that chloroplasts have a different diameter and sedimentation coefficient than other organelles, they are separated by centrifugation in a graded manner. The homogenate obtained by grinding the leaves is filtered and centrifuged to prepare chloroplasts. The periplasm of chloroplasts is relatively fragile, and the isolation of chloroplasts should be carried out in isotonic buffer solution at a temperature of 0~4 ℃. The viability of chloroplasts decreases as the time of isolation increases, therefore, the isolation should be completed in as short a time as possible.
Materials and Instruments
Material: fresh leaves of spinach or other green plants. Move The basic procedure for the isolation and preparation of plant chloroplasts can be divided into the following steps: 1. Select robust spinach leaves, preferably grown under several consecutive sunny days, wash and remove the petiole and midrib. 2. Take 10 g of cooled spinach leaves, tear them and put them into a mortar, and add 20 mL of 0.35 mol・L-1 NaCl, 2 mL of 0.01 mol・L-1 Tris buffer (or separation medium) and a small amount of quartz sand. Grind rapidly by hand for 30~60 s. Be careful not to use too much force, and do not grind too finely, as long as the leaf blade is ground into small pieces, and then filter the homogenate through 4 layers of new gauze. 3. Put the filtrate into two pre-cooled centrifugal tubes, balanced by a balance, and then centrifuged with a centrifuge at 1,000 r/min-1 for 2 min, and discard the precipitate. 4. 4. centrifuge the supernatant at 3,000 r・mir-1 for 5 min, discard the supernatant, and the precipitate is chloroplast. 5. Divide the precipitate into two parts and suspend them in 10 mL each of 0.35 mol・L-1 NaCl solution and 0. 035・L-1 NaCl solution, so that the chloroplasts are in isotonic solution and hypotonic solution respectively, and then complete chloroplasts and fragmented chloroplasts can be obtained. 6. Pipette a small amount of complete chloroplasts or broken chloroplasts suspension, add a small amount of measurement medium Ⅱ dilution, placed under a microscope (400 ~ 600 times), observe the morphology of chloroplasts. Caveat 1. Grind the leaf blades quickly and centrifuge them rapidly. 2. When preparing chloroplast suspensions, add the suspension medium slowly in order to maintain the integrity of the chloroplasts. For more product details, please visit Aladdin Scientific website.
Reagents:
Separation medium containing 0.33 mol・L
-1
sorbitol, 50 mmol ・L
-1
Tris-HCl (or Tricine) pH 7.6, 5 mmol・L -1
-1
MgCl
2, 10 mmol・L-1 MgCl
MgCl2, 10 mmol・L
-1 MgCl2, 10 mmol・L -1 MgCl2, 10 mmol・L -1
NaCL, 2 mmol・L
-1
EDTA-Na
2
NaCl, 2 mmol・L-1 EDTA-Na2, 2 mmol・L-1 EDTA-Na2
-1
sodium isoascorbate.
Formulation:
Weigh 60 g sorbitol, 6.06 g Trisa, 1 g MgCl
2
・6H
2
O, 0.6 g NaCl, 0.77 g ED1A-Na
NaCl, 0.77 g ED1A-Na2
0.4 g sodium isoascorbate, dissolved with 1 mol・L
1 mol・L-1
After dissolution, the pH was adjusted to 7.6 with 1 mol・L -1 HCI, and the volume was fixed to 1 000 mL.
Determination medium Ⅰ: containing 0.66 mol・L
-1
Sorbitol, 2 mmol・L
-1
MgCl
2
2 mmol・L
-1
MnCl
MnCl2
4 mmol・L
-1
EDTA-Na
MnCl2 ・4 mmol・L-1 EDTA-Na2
10 mmol・L-1 EDTA-Na 2
-Sodium pyrophosphate. 100 mmol・L
Sodium pyrophosphate, 100 mmol・L
-1
Tris-HCl pH 7.60.
Method of Preparation:
Weigh 60 g sorbitol, 0.2 g MgCl
2
・6H
2
O, 0.2 g MnCl
2
・4H
2
O, 0.75 g EDTA-Na
2
2.23 g Na
4
P
2
O
7
・10H
2
O, 6.06 g Tris, dissolved with 1 mol・L
-1 mol・L
After dissolution, adjust the pH to 7.6 with 1 mol・L-1 HCI, and then volume to 500 mL.
Measurement medium Ⅱ: Dilute the measurement medium Ⅰ by 1 times.
Separation medium can be used: 0.35 mol・L -1 HCI
-1
NaCl + 0.01 mol・L
0.01 mol・L-1 NaCl + 0.01 mol・L-1
Tris pH 7.6 buffer.
Equipment:
① Refrigerator;
② Centrifuge;
③ Balance;
④ Microscope;
⑤ pH meter;
⑤ pH meter. ⑥ Mantle;
⑦ Measuring cylinder;
⑧ Pipette;
⑨ Centrifuge tube;
⑩ skimmed gauze, etc.
Separation vessels should be pre-cooled at 0 °C.