Experiment on the isolation of chloroplasts from pea tissue
Experiment on the isolation of chloroplasts from pea tissue
This experiment is based on the "Guide to Cellular Experiments", translated by Huang Peitang et al.
Operation method
chloroplast separation
Materials and Instruments
Leaf tissue Move 1. Preparation of Percoll gradients For more product details, please visit Aladdin Scientific website.
PBF-Percoll solution Sorbitol BSA HEPES-KOH EDTA
Polycarbonate centrifuge tubes
(1) Add 25 ml of 50% PBF-Percoll solution to each of two 50 ml polycarbonate centrifuge tubes.
50% PBF-Percoll
0.33 mol/L sorbitol
50 mmol/L HEPES-KOH, pH 8.0
2 mmol/L EDTA
1 mmol/L MgCl2
1 mmol/L MnCI2
50% Percoll (v/v)
3% PEG 6000 (w/v)
1% BSA (fatty acid free, e.g. Sigma's A7030)
1% Ficoll (w/v)
(2) Centrifuge at 29,000 g (15,600 r/min, SS34 turn head; Sorvall) for 15 min to form a Pencoll gradient.
The resulting gradient can be left at 4°C overnight.
2. Tissue homogenization
(1) Harvest leaf tissue with scissors (about 100 g of leaf tissue can be obtained from one flat plate by culturing as described above), cut into pieces and transfer to a 1 L plastic beaker.
(2) Add ice-cold grinding suspension buffer. Grind the leaves with a Polytron homogenizer (Model PTA 10/35 with PTA-36/2 mol/L generator) in a room at 4°C for 6 to 7 cycles of two to three 5-second pulses.
Grind suspension (GR) buffer:
2 mmol/L EDTA sodium salt, pH 8.0
1 mmol/L MgCl2
1 mmol/L MnCl2
50 mmol/L HEPES-KOH, pH 8.0
0.33 mol/L sorbitol
0.5 g/L BSA ( fatty acid free)
5 mmol/L Ascorbic acid sodium salt (added before use)
(3) Filter the homogenate through two layers of Mirab (Calbiochem ). Pour the filtrate into two 250 ml centrifuge bottles.
3. Centrifuge the homogenate at 2600 g (Sorvall GSA turntable 4000 r/min) for 5 minutes.
4. Carefully decant the supernatant. Add 2 ml GR Buffer. Use a small camel hair brush to gently loosen the chloroplasts from the inside of the bottle. Vibrate on ice in a rotary shaker for 5-10 minutes to break up the clumps.
5. Add the resuspended chloroplasts to the Percoll gradient. centrifuge at 10500 g (8000 r/min on a Sorvall HB-4 turntable) for 10 minutes.
6. Draw off the gradient media until the band is at the bottom. Transfer the chloroplasts from each tube to a 50 ml centrifuge tube and dilute to 40 ml with 1x SH. Centrifuge at 2400 g for 2 minutes (SS34 turntable at 4500 r/min).
7. Resuspend the precipitate in a small amount of 1x SH using a camel hair brush. Pour all chloroplasts into a centrifuge tube, dilute to 40 ml, and centrifuge at 2400 g for 2 min.
8. Resuspend the precipitate in 1 to 2 ml of 1 x SH.
9. Determine the chloroplast yield by measuring chlorophyll.
(1) Mix 5 μl of chloroplast suspension with 995 μl of 80% acetone in a microcentrifuge tube.
(2) Centrifuge at 13,000 g for 2 minutes.
(3) Detect the light absorption values at 663 nm and 645 nm.
(4) Amount of chlorophyll (mg/ml) = (8.02 A663 + 20.2 A645 )/5
10. Long-term preservation of chloroplasts in liquid nitrogen.
(1) Immediately after isolation of the chloroplasts, the final precipitate was resuspended to a chlorophyll concentration of 3-5 mg/ml using 1x SH containing 20% DMSO.
(2) Place on ice for 10 minutes.
(3) Pipette one portion of each 1 ml into a microcentrifuge tube, freeze quickly, and store in liquid nitrogen.
11. Recovery of frozen intact chloroplasts.
(1) Thaw frozen chloroplasts at room temperature. Dilute with 1x SH to a chlorophyll concentration of 1 mg/ml.
(2) Aspirate 1 to 2 ml of 1x SH buffer containing 35% Percoll into a 15 ml centrifuge tube. Add the melted chloroplasts to it. centrifuge at 13000 g (Sorvall HB-4 turntable, 10000 r/min) for 3 minutes. Discard the supernatant and wash the precipitate with 1x SH.