Experiments on the preparation of crude microsomes from tissue culture cells
Experiments on the preparation of crude microsomes from tissue culture cells
This experiment is based on the "Guide to Cellular Experiments", translated by Huang Peitang et al.
Operation method
Preparation of crude microsomes from tissue culture cells
Materials and Instruments
Cells Move 1. Add cycloheximide (dissolved in distilled water at a storage concentration of 0.1 mol/L and stored at -20°C. If it does not dissolve, try heating to 40°C to help it dissolve) (final concentration 10 μmol/L) and hold at 37°C for 5-10 minutes. If it is not soluble, try to heat it up to 40℃ to help it to dissolve) (final concentration 10 μmol/L), and hold it at 37℃ for 5~10 minutes. For more product details, please visit Aladdin Scientific website.
Cycloheximide Homogenization Buffer
Petri Dish
2. Transfer the dish from 37℃ to a cold room, remove the medium immediately, wash the cells three times with ice-cold PBS containing the same concentration of cycloheximide as the medium, and place the dish upside down on a paper towel for a few minutes with occasional tapping. Wipe off any residual PBS on the side walls with Kimwipe tissue.
3. Add 0.7 ml of HM containing 2~4 units/ml RNase-IN (Boehringer Mannheim, Indianapolis, Indiana), and gently swirl the dish so that the HM, covers the bottom of the dish.
Homogenization buffer (HM):
10 mmol/L HEPES-KOH buffer, pH 7.5
10 mmol/L KCl
1 mmol/L MgCl2
1 mmol/L DTT
0.5 mmol/L PMSF
4. Hold the Petri dish at an angle, wet a rubber or plastic starch broom in the HM and scrape the cells from one side of the dish to the other in the same direction.
5. Suspend the scraped cells in the HM and concentrate them on one side of the dish, wet the cell broom and scrape the cells from the rest of the dish in the same way until the whole dish is scraped (the surface of the dish becomes smooth after the cells are scraped away). Wet the inner surface of the glass pipette with fresh HM (see step 4) and transfer the cell suspension into a small tightly matched Dounce homogenizer (Kontes).
6. Add 0.7 ml of fresh HM to the dish from which the cells have been scraped off, scrape the bottom of the dish with a cell precipitation broom, transfer the washings to another dish, scrape the cells in the same way, and concentrate the cell suspension into a Dounce homogenizer.
7. When the cell suspensions in two or three dishes have been pooled together, homogenize as follows:
(1) Hold the tube of the Dounce homogenizer at a slight angle on a table.
(2) Slowly insert the glass mortar and pestle until the head of the mortar and pestle is in contact with the surface of the cell suspension, and push the mortar and pestle up and down vigorously along one side of the tube.
(3) Repeat step (2) for a total of 15-20 pestle strokes in one upward and one downward motion.
8. Immediately after homogenization of the cells, transfer the homogenate to a test tube with a glass pipette and mix with 2.5 mol/L sucrose by 1/10 volume.
9. Repeat steps 3 to 8 until all cells in the dish have been homogenized.
10. Measure the volume of homogenate.
11. Centrifuge at 2100 r/min for 3 minutes in a Sorvall HB-4 turntable (700 g), increase speed to 6500 r/min (7000 g) and centrifuge for 10 minutes.
12. Transfer the supernatant (PMS) to a graduated cylinder and add 2.5 mol/L sucrose by 0.9 volume for a final sucrose-PMS mixture of 1.33 mol/L or 2.5 mol/L sucrose by 2.2 volume for a final sucrose-PMS mixture of 1.8 mol/L.
13. Prepare the following graded gradient (bottom to top) in a SW41 turn-head centrifuge tube:
Gradient a:
1.5 ml 1.8 mol/L sucrose-HM
1.5 ml 1.5 moI/L sucrose-HM containing 2~4 units/ml RNase-IN
5 ml sucrose-PMS mixture
1.5 ml 1.0 mol/L sucrose-HM
1.5 ml 0.6 mol/L sucrose-HM
1 ml 0.25 mol/L sucrose-HM
Gradient b:
5 ml of sucrose-PMS mixture
1.5 ml of 1.5 mol/L sucrose-HM containing 2 to 4 units/ml RNase-IN
1.5 ml 1.3 mol/L sucrose-HM with 2-4 units/ml RNase-IN
1.5 ml of 1.0 mol/L sucrose-HM
1.5 ml 0.6 mol/L sucrose-HM
1 ml 0.25 mol/L sucrose-HM
14. Centrifuge in a SW41 turntable at 41000 r/min (286500 g max) at 4°C for 16 hours (gradient a) or at least 5 hours (gradient b).
15. Collect the material formed at the interface of the 1.5 mol/L and 1.8 mol/L sucrose layers, which is considered to be the heavy coarse microsomal fraction.
16. Concentrate the microsomes according to the purpose of the experiment by one of the following methods.
(1) To obtain a good isolation profile of the polysomes
(1) Dilute a portion of the microsomes 100 times with 0.1% SDS solution and measure the absorption values at 260 nm and 280 nm.
② Estimate whether 100 μl of microsomes contains enough ribosomes to show the distribution of polyribosomes in a sucrose density gradient.
(iii) Dilute the sample 5-fold or 7-fold with HM and analyze in a sucrose linear density gradient with a SW41 turntable.
(2) To collect the precipitate
① Dilute the sample 3-fold or 5-fold with HM.
① Dilute the sample 3 times with HM or 5 times with HM. ② Centrifuge the sample in a Beckman Ti60 or Ti50 turntable at 40,000 r/min (max. 160,000 g) for 60-80 minutes, depending on the volume of the sample.
(3) For in vitro translation and reconstruction experiments
① Dilute the sample 3-fold or 5-fold with HM containing RNase inhibitor.
① Dilute the sample with a 3-fold or 5-fold dilution of HM containing RNase inhibitor. ② Add the diluted sample to a 1.8 mol/L sucrose cushion solution and centrifuge the sample for 60 min at 35,000 r/min (max. 210,000 g) in a SW41 or SW60 turntable (the choice of turntable depends on the volume of the sample).
(iii) Collect the microsomal membrane bands formed in the upper portion of the cushion solution and further concentrate to a smaller volume using the SW60 head if necessary.