Preparation of nuclear and cytoplasmic S100 extracts of HeLa cells for in vitro splicing reaction assay
Cytosolic extracts can be used for splicing of precursor mRNAs. The S100 extract, although it includes many splicing factors, does not have splicing activity because it contains only some of the SR proteins necessary for splicing, although effective splicing can be carried out by supplementation of one or more SR proteins in this extract. This experiment is from the "RNA Laboratory Guidebook", edited by Xiaofei Zheng.
Operation method
Preparation of nuclear and cytoplasmic S100 extracts of HeLa cells for in vitro splicing reaction assay
Principle
Cytosolic extracts can be used for splicing of precursor mRNAs, and although the S100 extract includes many splicing factors, it is not splicing active because it contains only some of the SR proteins necessary for splicing, although the extract can be efficiently spliced when supplemented with one or more SR proteins.
Materials and Instruments
HeLa cells Move -Materials and equipment For more product details, please visit Aladdin Scientific website.
DTT Phenylmethylsulfonyl Fluoride PBS Buffer
Eagle Basic Medium for Suspension Cultures Glass Hook and Loop Dialysis Bags
All reagents should be prepared in high quality autoclaved water such as Mill Q ( Millipore, Bedford, MA, USA) or double swallow distilled water. All components used for cell culture should be autoclaved, and heat-resistant materials should be filtered with a 0.22 μm filter to remove bacteria.
1. HeLa cell suspension culture
(1) Medium: Joklik's modified Eagle Basic Medium for Suspension Cultures (ICN Pharmaceuticals, Inc.), or an equivalent medium, which is a powdered medium containing all the necessary ingredients except for haematocrit. Dissolve the powder in Milli Q water, filter and sterilize to make a 5X stock solution, make a working solution from filtered sterilized water and add calf serum to a final concentration of 50 ml/L, pH approximately 7.0.
(2) HeLa cells: Use cell lines suitable for growth in suspension medium, such as S-3.
2. Preparation of extracts
Reagent (1) and (2) stocks should be stored at -20°C and solutions (3)~(6) added before use.
(1) 1 mol/L DTT.
(2) 20 mg/ml (115 mmol/L ) phenylmethylsulfonyl fluoride (PMSF), dissolved in ethanol.
(3) PBS: 137 mmol/L NaCl, 2.7 mmol/L KCl, 8 mmol/L Na2HPO4, 1.5 mmol/L KH2PO4, 0.5 mmol/L MgCl2.
(4) Buffer A: 10 mmol/L HEPES-KOH ( pH 8.0), 10 mmoI/L KCl, 1.5 mmol/L MgCl2, 1 mmol/L DTT.
(5) Buffer B: 0.3 mol/L HEPES-KOH ( pH 8.0), 1.4 mol/L KCl, 30 mmol/L MgCl2.
(6) Buffer C: 20 mmol/L HEPES-KOH ( pH 8.0), 0.6 mol/L KCl, 1.5 mmol/L MgCl2, 0.2 mmol/L EDTA, 25% (V/V) glycerol, 0.5 mmol/L PMSF, 1 mmol/L DTT.
(7) Buffer D: 20 mmol/L HEPES-KOH ( pH 8.0), 100 mmol/L KCl, 0.2 mmol/L EDTA, 20% ( V/V) glycerol, 0.5 mmol/L PMSF, 1 mmol/L DTT.
(8) 40 ml Dounce glass hook and loop vessel with loose pestle and mortar.
(9) A dialysis bag with a cut-off molecular mass of 12000-14000 Da.
Methods
1. HeLa cell suspension culture
Cells were cultured at 37℃ in a rotating culture flask at a cell density of 2X105~5X105. The cell multiplication time was about 24 hours and the cell density could be counted by a blood cell counter.
2. Preparation of HeLa cell nuclear extracts
(1) Take HeLa cells in logarithmic growth phase ( 4X105~6X105/ml, not more than 1X106/ml ) and centrifuge at 1800 g for 10 min with low speed centrifuge to collect the cells.
(2) Determine the volume of cellular precipitate (PCV ) by gently resuspending the cells with ice-cold PBS and centrifuging again. All of the latter steps should be performed on ice or in a cold room, and all centrifugation steps should be performed at 4 °C.
(3) Gently resuspend the cell precipitate in 5x PCV of Buffer A and place on ice for 10 min to allow the cells to swell in hypotonic buffer.
(4) Centrifuge at 1800 g for 10 min to precipitate the cells. Resuspend the cells in Buffer A with 2x PCV. The volume of cells precipitated will be doubled due to swelling and the original PCV will be used in the calculation.
(5) Homogenize approximately 10 times with a Dounce glass homogenizer. The extent of cell lysis is determined by hematocrit using Taipan blue staining, aiming for 90% cell lysis, but do not homogenize more than necessary. Note that HeLa cells have large nuclei and are not much smaller than intact cells.
(6) The lysate is transferred to a high-speed centrifuge tube and centrifuged at 1200 g for 10 min. The supernatant is carefully pipetted and added to a graduated cylinder to prepare the S100 extract.
(7) Centrifuge at 33,000 g for 20 min in the same tube and carefully discard the supernatant.
(8) The concentrated nucleus volume (PNV) obtained from 12 liters of HeLa cell culture is typically 9-15 ml. Add 2 ml of Buffer C, spin to separate the precipitate from the centrifuge tube but do not disperse the precipitate, transfer the precipitate and liquid to a graduated disposable tube, measure the volume, and subtract 2 ml for an estimated PNV, add more Buffer C (containing 0.6 mol/L KCl) to make the final KCl volume. KCl) to give a final concentration of KCl of 0.24 mol/L, which should range from 0.20 to 0.25 mol/L.
(9) Transfer the precipitate and buffer (do not resuspend the precipitate yet or the solution will become viscous and difficult to transfer without loss) to a clean 40 ml glass Bounce homogenizer and homogenize several times with a loose mortar and pestle to keep it in suspension. Ten homogenizations should be sufficient to prepare a homogenous suspension.
(10) Transfer the homogenate to one or more high-speed centrifuge tubes with screw caps and mix with gentle shaking for 30-45 min.
(11) Precipitate the salt-washed nuclei by high-speed centrifugation (~33,000 g for 30 min) and carefully transfer the supernatant to a disposable tube to avoid contamination of the precipitate.
(12) Dialyze the supernatant twice with Buffer D (usually 1~2 L). One time overnight and the other time for at least 4 h. The supernatant should be dialyzed in Buffer D.
(13) High-speed centrifugation at 33,000 g for 20 min removes the precipitate formed during dialysis and carefully transfers the supernatant (nuclear extract) to a disposable test tube.
(14) Dispense the extract into microcentrifuge tubes at 0.1~1 ml. Quickly freeze the tubes in liquid nitrogen and store at -70°C or lower.
3. Preparation of HeLa cell cytoplasmic S100 extracts
While awaiting centrifugation in step (7) of Procedure 1, "Preparation of HeLa Cell Nuclear Extracts", the supernatant from step (6) is processed to yield an S100 extract, which consists of the cytoplasmic component and many, but not all, of the nuclear component, and which is extracted in Hypotonic Buffer A. The S100 extract is prepared from the cytoplasm of HeLa cells in the following manner
(1) Measure the volume of the supernatant, add 0.11 times the volume of Buffer B, and mix gently.
(2) Centrifuge at 100,000 g for 1 h in an ultracentrifuge tube.
(3) Transfer the supernatant into a disposable tube.
(4) Dialyze twice, once overnight and again for at least 4 h, with Buffer D. The volume of the extract is significantly reduced during dialysis because the extract is concentrated by the glycerol contained in Buffer D. The extract should be dialyzed twice, once overnight and again for at least 4 h. The volume of the extract should be reduced by the glycerol contained in Buffer D.
(5) Insoluble material was removed by high-speed centrifugation at 33,000 g for 20 min.
(6) Dispense 0.1-1 ml of extract into microcentrifuge tubes, freeze rapidly and store as described in step (14) of Procedure 3, "Preparation of Nucleus Extracts from HeLa Cells".12 Liters of culture will yield 40-55 ml of cytoplasmic S100 extracts with a total protein concentration of 10-15 mg/ml. Extract activity can be maintained for several years.