Purification experiments of U series small ribonucleoprotein particles
Purification experiments of U series small ribonucleoprotein particles
The splicosome is the catalytic unit for excising introns from primary transcripts in eukaryotes. The splicosome contains four small nuclear ribonucleoprotein particles (snRNPs), U1, U2, U4/U6, U5 snRNP, and a large number of non-snRNP proteins. This experiment is from the "RNA Laboratory Guidebook", edited by Xiaofei Zheng.
Operation method
Purification experiments of U series small ribonucleoprotein particles
Principle
The splicosome is the catalytic unit for excising introns from primary transcripts in eukaryotes. The splicosome contains four small nuclear ribonucleoprotein particles (snRNPs), U1, U2, U4/U6, U5 snRNP, and a large number of non-snRNP proteins.
Materials and Instruments
HeLa Cells Move -Materials and equipment For more product details, please visit Aladdin Scientific website.
PBS-Earle Buffer Phosphate Buffer
Dounce Screeder Mortar and Pestle S Immunoaffinity Chromatography Columns Anion Exchange Columns
1. Preparation of HeLa cell nucleus extracts
(1) HeLa cells in suspension culture.
(2) PBS-Earle: 130 mmol/L NaCl, 20 mmol/L K2HPO4 / KH2PO4 ( pH 7.4).
(3) Buffer A: 10 mmol/L HEPES-KOH (pH 8.0), 10 mmoI/L KCl, 1.5 mmol/L MgCl2, 0.5 mmol/L 1,4 disulfide erythritol ( DTE ).
(4) Buffer C: 20 mmol/L HEPES-KOH ( pH 8.0), 420 mmol/L NaCl, 1.5 mmol/L MgCl2, 0.5 mmol/L DTE, 0.5 mmol/L phenylmethylsulfonylfluoride (PMSF), 0.2 mmol/L EDTA (pH 8.0), 25% ( V/V) glycerol.
(5) Buffer G: 20 mmol/L HEPES-KOH ( pH 8.0), 150 mmol/L KCl, 1.5 mmol/L MgCl2, 0.5 mmol/L DTE, 0.5 mol/L PMSF, 5% ( V/V) glycerol.
(6) 40 ml Dounce homogenizer and a properly loosened S-shaped mortar and pestle.
2. Glycerol gradient centrifugation
(1) Buffer C: 20 mmol/L HEFES-KOH ( pH 8.0), 420 mmol/L NaCl, 1.5 mmoL/L MgCl2, 0.5 mmol/L DTE, 0.5 mmol/L PMSF, 0.2 mmol/L EDTA (pH 8.0).
(2) Buffer G: 20 mmol/L HEPES-KOH ( pH 8.0), 150 mmol KCl, 1.5 mmol/L MgCl2, 0.5 mmol/L DTE, 0.5 mmol/L PMSF, 5% (V/V) glycerol.
3. U-series snRNP was purified by immunoaffinity chromatography using anti-m3G.
(1) Anti-M3G Immunoaffinity Chromatography Column: Approximately 7 mg of anti-M3G IgG (Euro-Diagnostica, Arnhem, The NetherUmds ) was bound to 1 ml of CNBr-activated Stpharose gel according to the standard method provided by Pharmacia.
(2) Buffer C: 20 mmol/L HEPES-KOH (pH 8.0), 420 mmol/L NaCl, 1.5 mmol/L MgCl2, 0.5 mmol/L DTE, 0.5 mmol/L phenylmethylsulfonylfluoride (PMSF), 0.2 mmol/L EDTA ( pH 8.0 ), 25% (V/V) glycerol.
(3) Buffer C ( low): Same composition as Buffer C except 5% glycerol was used.
(4) Buffer solution: Same composition as Buffer C (low) except NaCl concentration is 250 mmol/L.
4. Purification of U1 snRNP by Mono Q chromatography
(1) Buffer Q-0: 20 mmol/L Tris-HCl (pH 7.0), 1.5 mmol/L MgCl2, 0.5 mmol/L DTE, and 0.5 mmol/L PMSF.
(2) Buffer Q-50: 50 mmol/L KCl was added to buffer Q-0.
(3) Buffer Q-1000: 1000 mmol/L KCl was added to Buffer Q-0.
(4) 1 ml Mono Q anion exchange column (Pharmacia).
5. [ U4/U6, U5 ] Purification of trimeric snRNP complexes
(1) Immunoaffinity column for H386 monoclonal IgG antibody (Euro-Diagnostica, Arnhem, The Netherlands).
(2) The H386 antibody epitope, a 32-peptide with the sequence DRDRERRRSHRSERERRRDRDKDRURDREHKR, can be synthesized by standard methods.
(3) Buffer G: 20 mmol/L HEPES KOH ( pH 8.0), 150 mmol/L KCl, 1.5 mmol/L MgCI2, 0.5 mmol/L DTE, 0.5 mmol/L PMSF, 3% ( V/V) glycerol.
(4) Phosphate buffer: 10 mmol/L sodium phosphate (pH 7.2 ).
6. Purification of 17S U2 snRNP
(1) Anti-m3G immunoaffinity chromatography column: as before.
(2) Buffer G: as before.
II. Methods
1. Preparation of HeLa cell nucleus extracts
(1) Cultivate HeLa S3 cells suspended at 37℃ in D MEM medium with 5% newborn calf serum, 50 μg/ml penicillin, 100 μg/ml streptomycin, and keep the cell density in logarithmic growth phase at 2. 5X105~5X105/ml. In order to get enough U snRNP, the cell number should reach 5X109 (equivalent to culturing 8~10 L cells). 10 L cells).
(2) Collect the cells by centrifugation at 1000 g for 10 min at 4℃.
(3) Resuspend every 109 cells with 20 ml PBS-Earle and collect the cells by centrifugation at 1000 g for 10 min.
(4) Estimate the volume of cellular precipitate and resuspend with 5 times the volume of Buffer A.
(5) Allow the cells to swell for 10 min, then centrifuge as in step (3) and resuspend with 2x volume of Buffer A.
(6) Homogenize 10 times with a Dounce homogenizer to break up the cells.
(7) Remove cytoplasm by centrifugation twice in succession with SorvalI SA1: rotor; 1000 g for 10 min then 25000 g for 20 min.
(8) Resuspend precipitate with Buffer C. Use 3 ml of Buffer C per 105 cells.
(9) Homogenize 10 times with a Dounce homogenizer to break up nuclei.
(10) Transfer the remaining suspension to a beaker and gently stir with a magnetic stirrer on ice for 30 min.
(11) Centrifuge at 25000 g for 30 min to remove the nuclear membrane.
(12) The remaining supernatant is the cytosolic extract obtained under high salt (420 mmol/L) conditions. The extract can be stored at -80°C after snap-freezing in liquid nitrogen.
To obtain splicing-active cytosolic extracts, dialyze a 100-fold volume of buffer G for 4-5 h to a salt concentration of 150 mmol/L. Cytosolic extracts prepared in this manner are splicing-active at 40% to 60% of the final reaction volume. 5x109 cells yield approximately 18 ml of low-salt cytosolic extract.
2. Glycerol gradient centrifugation
Depending on the size of the snRNP, different types of snRNP can be separated by glycerol density gradient centrifugation. initial graded separation of the cytosolic extracts by density gradient centrifugation significantly improves the purity of the snRNP obtained. Lower salt concentrations should be used for the initial separation of 17S U2 snRNP and 25S [ U4/U6, U5 ] poly-snRNP by density gradient centrifugation.
(1) Prepare a 10%-30% linear glycerol density gradient with Buffer C (high salt gradient) or Buffer G (low salt gradient) in a sterile SW 28 tube at room temperature.
(2) Allow to equilibrate within each density gradient by standing at 4°C for at least 1 h (preferably overnight).
(3) Carefully add the cytosolic extract to the density gradient using a pipette, with a 30 ml SW 28 density gradient column capable of holding 8 ml of extract.
(4) Centrifuge at 2700 r/min ( 105 g) with low acceleration for 17 h. Terminate centrifugation without braking.
(5) Collect the gradients with an automatic graduation device or manually with an Eppendorf pipette from top to bottom in 1.5 ml portions.
(6) Take 1/10 of each fraction, phenol: chloroform extraction followed by ethanol/sodium acetate precipitation to obtain RNA. analyze the U-series snRNP of each fraction by gel electrophoresis and by standard silver staining.
3. Purification of U-series snRNP by immunoaffinity chromatography with anti-m3G
(1) Equilibrate the immunoaffinity column with anti-m3G by washing the column with 5 times the volume of Buffer C. At least 5 ml of the column volume should be used. A minimum column volume of 5 ml should be used to effectively bind the U snRNP in 15 ml of cytosolic extract.
(2) Pass the cytosolic extract obtained under high salt conditions through the affinity chromatography column at 1.5 ml/h.
(3) Elute the non-specifically bound components with 6 times the column volume of buffer C (low).
(4) Specifically bound U snRNP was eluted with 2 times the column volume of 15 mmol/L nucleoside. m3G can be dissolved in high salt buffer [ Buffer C ( low)] or in a medium concentration of salt solution (Buffer F ). If Buffer F is used, U4, U5, U6 snRNP will be eluted as [ U4/U6, U5 ] poly-snRNP complexes, and about 4 mg of U snRNP can be isolated from 15 ml of cytosolic extract. snRNP can be stored at -80°C after snap-freezing in liquid nitrogen.
(5) The antibody-bound m3G can be washed off by washing the column with 6 mol/L urea [dissolved in buffer C (low)] at 1x column volume.
(6) Washing the column with 20 times the column volume of Buffer C (low) will regenerate the affinity chromatography column, but for long term storage a final concentration of 0.02% (V/V) NaN4 should be added.
4. Purification of U1 snRNP by Mono Q chromatography
(1) Wash the chromatography system with 20x volume of buffer Q-1000.
(2) Wash the column with the same volume of Buffer Q-50 as in step 1 and equilibrate. Measure the light absorption at 280 nm and use this value as the zero point.
(3) Dilute U snRNP obtained by anti-m3G immunoaffinity chromatography with Buffer Q-0 so that the concentration of monovalent ions is less than 200 mmol/L.
(4) Load U snRNP ( 1~40 mg ) onto a Mono Q column at a flow rate of 2 ml/min. pressure should not exceed 3.0 MPa.
(5) Wash the column with Buffer Q-50 until the light absorption value is zero (usually twice the column volume).
(6) Elute snRNP at a flow rate of 1 ml/min and control the KCl concentration gradient by programming the FPLC controller. Start with a KCI concentration of 50 mmol/L (Buffer Q 50), then increase Buffer Q-1000 by 5.4% per minute for 4 min, then 1% per minute for 30 min, then 4.2% per minute for 10 min. The final wash with Buffer Q-1000 was performed for 4 min. 1 ml of each was collected during the elution process. elution of U1 snRNP reached its first peak at a KCl concentration of 350-370 mmol/L. The elution of U1 snRNP was performed at a KCl concentration of 1.4% for 4 min, then 1% for 30 min, then 4.2% for 10 min.
(7) The concentration of U1 snRNP in each collection was determined by measuring the light absorption at 280 nm (I A280 unit equivalent to 0.35 mg/ml), and the RNA and protein components were analyzed by PAGE.
(8) 20S U5 snRNP contaminated with 12S U1 snRNP can be further separated by glycerol density gradient centrifugation.
5. purification of [ U4/U6, U5 ] dimeric snRNP complexes
Two methods can be used to isolate the [ U4/U6, U5 ] trimeric snRNP complex. The first method is to separate the total snRNP obtained by cap-specific antibody immunoaffinity chromatography by glycerol gradient centrifugation, which is faster and more productive, but the purity of the trimeric snRNP is lower. The second method was immunoaffinity purification with the H386 antibody, which was prepared using the U1 snRNP-specific 70 kDa protein, but it also strongly cross-reacted with a 100 kDa protein in the [ U4/U6, U5 ] trimeric snRNP complex. The [ U4/U6, U5 ] trimeric snRNP obtained by this method is relatively pure, but it is more cumbersome and less productive than the first method. The second method will be briefly described below.
(1) Combine the 25S nuclear extracts separated by glycerol density gradient centrifugation and pass them through an H386 immunoaffinity column at a flow rate of 1 ml/min. A 2 ml column volume can contain up to 7 ml of nuclear extracts, which corresponds to 4 collections containing approximately 100~150 μg U snRNP.
(2) Wash out non-specifically bound components with 20 times the column volume of Buffer G.
(3) Elute specifically bound U snRNP with a 5-fold column volume of Buffer G containing 0.01 mmol/L competitive 32 peptide relative to the major antigenic epitope of H386. 400 μl of each collection is taken, and 1/10 of it is analyzed for RNA and protein fractions by standard methods.
(4) If necessary, trace amounts of 12S U1 snRNP co-eluting with 25S [ U4/U6, U5 ] trimeric snRNP can be separated by glycerol gradient centrifugation as described previously.
(5) Elute with 5 times the column volume of phosphate buffer (10 mmol/L sodium phosphate, pH 7.2) followed by the same volume of the same buffer containing 3.5 mol/L MgCl2 to elute the antibody-bound peptide.
(6) Regenerate the affinity column by washing with 10 times the column volume of buffer G. For long-term storage, add a final buffer. For long-term storage add NaN3 at a final concentration of 0.02% (V/V).
6. Purification of 17S U2 snRNP
It is very difficult to obtain 17S U2 snRNP with the complete protein fraction because most of the U2-specific proteins are separated from the U2 snRNP when the salt concentration exceeds 200 mmol/L, and it is also difficult for the antibody to bind to the m3G cap of the U2 RNA in 17S U2 snRNP. Therefore, prior to the immunoaffinity separation of the 17S U2 snRNP with the cap-specific conjugate antibody, all experimental operations should be performed at low temperature and with a high concentration of NaN3. All experimental manipulations should be performed at low ionic strength, and most of the snRNP with good exposed cap structure should be removed prior to purification. when isolating from cytosolic extracts, preisolates of 17S U2 snRNP should be obtained by centrifugation with a glycerol gradient.
(1) Combine the fractions obtained by glycerol density gradient centrifugation. Remove small amounts of 12S U1 snRNP, U5 snRNP, and [ U4/U6, U5 ] trimeric snRNP with an H386 immunoaffinity chromatography column.
(2) The effluent containing 17S U2 snRNP is loaded onto an anti-m3G immunoaffinity chromatography column.
(3) Elute the non-specifically bound material with 20 times the column volume of Buffer G.
(4) Elute 17S U2 snRNP with m3G as described in Procedure 2, Glycerol Gradient Centrifugation, and analyze.
(5) Regenerate the affinity column as described in step (3) of Procedure 2, "Glycerol Gradient Centrifugation".