Experiments on the preparation of nuclear extracts from HeLa cells
Experiments on the preparation of nuclear extracts from HeLa cells
In this experiment, nuclear extracts were prepared from HeLa cells essentially as described by Dignam et al. (1983). This basic method is probably the most commonly used method for preparing factors for in vitro transcription and DNA binding experiments. This experiment was derived from the Laboratory Guide for Protein Purification and Identification by Hauchu Zhu.
Operation method
Experiments on the preparation of nuclear extracts from HeLa cells
Materials and Instruments
Glycerol Liquid Nitrogen MgCl2-6 H2O Ammonium Sulfate HeLa Cell Phosphate Buffered Salt Solution (PBS) Buffer G Buffer H Buffer D TM Buffer + 0.1 ml L KCl Move Materials For more product details, please visit Aladdin Scientific website.
HeLa cells (see below for culture and storage conditions. We have also successfully used HeLa cells prepared and frozen by CellexBiosciences)
Glycerol
Liquid Nitrogen
MgCl2-6H2O
Ammonium Sulfate
Reagents
Phosphate Buffered Salt Solution (PBS)
Buffer G
Buffer H
Buffer D
TM Buffer + 0.1 ml/L KCl
(For the recipe, see "Preparation of Reagents", pp. 131-138.)
Operating Procedures
HeLa cell culture and preservation (12-L scale)
1) HeLa cells are grown logarithmically to a density of 5X105 cells/ml, and the eluments are collected gently to avoid lysis.
2) Cells are washed once with PBS and collected again.
3) 2 ml of buffer G are added to each liter of original culture (i.e., 24 ml of buffer are added to the cells from the 12-L culture, and glycerol is added to the final concentration of glycerol. Glycerol was added to a final concentration of 20% glycerol (v/v). For example, a volume of approximately 18 ml of cells can be obtained from 12L of culture medium, and a total volume of 42 ml is obtained by adding 24 ml of buffer G. However, the glycerol concentration is only 17.1% due to the volume occupied by the cells, and 1.5 ml of glycerol must be added to achieve a final glycerol concentration of 20%.
4) Freeze the cells in liquid nitrogen, and store them at -80°C. HeLa cell nuclei are prepared on a 12-L scale (12-L). Scale)
All solutions should be kept at 4°C and all operations should be carried out at 4°C unless otherwise stated.
1) Thaw HeLa cells (from 12L of culture medium with a cell density of approximately 5X105 cells/ml) in water at room temperature. The cells should be thawed as quickly as possible, but should not be thawed above 4°C. Add PBS containing lg/L MgCl2-6H20/L to a final volume of 200 ml, and then centrifuge the cells for 10 min at 3000 r/min with a BeckmanGSA turn-table.
2) Wash the cells with 200 ml of 1XPBS containing 1 g/L MgCl2-6 H20, and then centrifuge the cells for 10 min at 3000 r/min with a BeckmanGSA turn-table. 3) Wash the cells with 200 ml of 1XPBS containing 1 g/L MgCl2-6 H20, and then centrifuge the cells for 10 min at 3000 r/min. 10 min, collect the cells.
3) Resuspend the cells in 60 ml of buffer H.
4) Incubate the cells on ice for 15~30 min.
5) Crush the swollen cells with Dounce's homogenizer (40 ml) and pestle and mortar and grind them for 20 times.
Note: It is important to operate quickly after cell lysis to minimize the loss of transcription factors due to leakage outside the nucleus. In fact, high concentrations of nuclear transcription factors are commonly found in the supernatant from the nuclei collected by centrifugation of cytoplasmic fractions. Therefore . Increasing the number of washes and centrifugations tends to reduce the yield of transcription factors.
6) Collect nuclei by centrifugation at 3500 r/min for 10 min using a Beckman SS-34 turntable. Nuclear extracts were prepared immediately. Approximately 12 g of nuclei are typically obtained from 12 L of Hela cell culture. HeLa Cell Shuttle Extract Preparation (12-L Scale)
All solutions should be maintained at 4°C and all manipulations should be carried out at 4°C, unless otherwise noted.
1) Take nuclei prepared from 12 L of HeLa cell culture (thaw one copy of frozen nuclei as described above or in cold water ( 4°C ).
2) Suspend nuclei in a 75 ml buffer of cell culture. cell nuclei suspended in 75 ml of Buffer D.
Note: 0.42mol/L KC1 in cuffing buffer can be replaced by 0.42mol/L NaCl.
3) Stir with a magnetic stirrer for 30 min.
4) Centrifuge at 100OOOOg for 1 h to precipitate cell debris.
Note: For this step, it is recommended to use a bucket-type rotor (e.g. BeckmanSW28) instead of a fixed-angle rotor. The latter can cause undesired shearing of the nuclei.
5) Measure the volume of supernatant (usually 70-75 ml).
Note: To prepare a standard extract for in vitro transcription experiments, the supernatant from a 100,000 g centrifugation can be dialyzed against TM buffer containing 0.1 mol/LKCl.6 Alternatively, ammonium sulfate precipitates can be dialyzed against TM buffer + 0.1 mol/LKC1, as described below. 0.42 mol/LKC1 extracts contain high levels of histone H1, which is a potential inhibitor of transcription. The 0.42 mol/LKC1 extract contained high levels of histone H1, which is a potential inhibitor of transcription (Crceton et al., 1991). Ammonium sulfate precipitation removes most of the histone II1, so it is advisable to precipitate 0.42 mol/LKC1 extract with ammonium sulfate before dialyzing the sample against TM buffer + 0.1 mol/LKC1.
6) Add 0.33 g of ammonium sulfate per ml of supernatant, and add it slowly over 5-10 min.
Note: Crush the ammonium sulfate crystals about 5~10 min before use. Ammonium sulfate powder is hygroscopic and moisture absorbing ammonium sulfate is difficult to weigh accurately. A coffee bean grinder is a good utensil for grinding ammonium sulphate, much better than a mortar and pestle.
7) After all the ammonium sulphate has been dissolved, the mixture is stirred with a magnetic stirrer for 30 min.
8) Centrifuge the protein for 15 min at high speed with a BeckmanSS-34 turntable at 15000 r/min.
9) Dissolve the precipitate in about 3.5 ml of TM buffer + 0.1 mol/ LKCl and allow the solution to settle. LKCl and make the final volume of the solution about 5 ml.
10) The resulting milky-white mixture was centrifuged at high speed for 10 min at 10,000 r/min with a Beckman SS-34 turntable to remove insoluble material.
11) The supernatant was generally milky-white in color, and it could be directly put on an S-300 column for gel filtration. The protein concentration is usually about 50 mg/ml, determined by Bradford's method (using bovine γ-globulin as a reference).
12) If necessary, the extract can be frozen in liquid nitrogen, and can be stored for months (or even years) at -80°C. The extract should be thawed as much as possible, and should be kept in the liquid nitrogen bath. It is important that the extract melts as quickly as possible. This can be done by placing the frozen tube in room temperature water and removing any ice that may have formed around the tube. After the extract has thawed, it is important to centrifuge it at high speed for 10 min at 10,000 r/min using a Beckman SS-34 head to remove insoluble material. Alternatively, the extract can be dialyzed against TM buffer + 0.1 mol/L KCl for a few hours until the conductivity of the sample is identical to that of the buffer, and the nuclei extract is ready to be used for in vitro transcription experiments.