Precursor mRNA ( pre-mRNA ) splicing factors The SR (serine/argininc rich) family of proteins is an important player in the assembly of splices onto precursor mRNAs.SR proteins are important splicing factors, and different members of the family can direct the selection of selective splice sites in vitro or in vivo. This experiment was derived from "RNA Lab Guidebook", edited by Xiaofei Zheng.
Operation method
Purification of splicing factor SR protein assay
Principle
Precursor mRNA ( pre-mRNA ) splicing factors The SR (serine/argininc rich) family of proteins is an important player in the assembly of the spliceosome onto precursor mRNAs.SR proteins are important splicing factors, and different members of the family can direct the selection of selective splice sites in vitro or in vivo.
Materials and Instruments
Ultrapure Ammonium Sulfate Move I. Materials and equipment For more product details, please visit Aladdin Scientific website.
SR Protein Extraction Buffer SR Dialysis Buffer Acrylamide Solution Protein Spiking Buffer Acetone Guanidine Hydrochloride Khomas Brilliant Blue Staining Solution
Large probe ultrasonograph Phase contrast microscope Centrifuge Centrifuge tubing Dialysis bag Polyacrylamide gel electrophoresis device
1. Purification of SR protein family from cells and tissues
(1) Low-speed centrifugation was used to collect cells cultured to mid-logarithmic growth, washed twice with PBS at 4°C, centrifuged to collect cells and stored at -80°C for later use. The following preparation experiments usually use 6X109~8X1011 HeLa cells or 4X1010~5X1015 Drosophila Kc cells. The cell precipitate is ground to a powder in liquid nitrogen, noting that the mortar should be pre-cooled on dry ice before adding the extraction buffer.
(2) Animal organs and tissues may be prepared as described below. Ensure that the tissues are stored in liquid nitrogen or dry ice within a few minutes of the animal's death. The tissues should be powdered in a mortar and pestle placed on dry ice or in liquid nitrogen, which should be added continuously during the grinding process, and the pulverized tissues can be stored at -80°C for later use.
(3) SR protein extraction buffer: 10 mmol/L HEPES-KOH (pH 7.6), 67 mmol/L KCl, 13 mmol/L NaCl, 10 mmol/L EDTA, 5 mmol/L DTT, 5 mmol/L KF, 5 mmol/L β-phosphoglycerol, 0.2 mmol/L PMSF, 500 ml was prepared. ml.
(4) SR dialysis buffer: 10 mmol/L HEPES-KOH (pH 7.6), 67 mmoI/L KCl, 13 mmol/L NaCl, 1 mmol/L EDTA, 2 mmol/L DTT, 5 mmol/L KF, 5 mmol/L β-phosphoglycerol, 0.2 mmol/L PMSF, and prepare 4 L. The dialysis buffer was used as the buffer for the dialysis.
(5) Buffer D: 20 mmol/L HEPES-KOH (pH 7.6), 5% (V/V) glycerol, 0.1 mol/L KCl, 0.2 mmol/L EDTA, 0.5 mmol/L PMSF, 0.5 mmol/L DTT, 50 ml was prepared.
(6) Large probe sonicator: Branson Ultrasound Crusher 450 or equivalent, 0.5 inch (1 inch = 2.54 cm ) diameter crushing probe. Tissue culture cells as well as grinded tissue are placed in a 600 ml beaker and broken in extraction buffer.
(7) Phase contrast microscope to monitor inoculation cell fragmentation during sonication.
(8) Centrifuge and centrifuge tubes: Backman J2 or equivalent centrifuge, JA-17 or equivalent rotor, 50 ml polypropylene centrifuge tubes with lids.
(9) Ultracentrifuge and tubes: Beckman L7 or equivalent centrifuge, SW-28 or equivalent large-capacity bucket rotor, and matching tubes.
(10) Ultrapure ammonium sulfate: carefully pulverized in a mortar and pestle before use.
(11) 90% Ammonium Sulfate SR Dialysis Buffer: 66.2 g ammonium sulfate dissolved in 100 ml SR dialysis buffer.
(12) MWCO (Molecular Weight Cutoff) 6000~8000 dialysis bag, the bag was boiled in 10 mmol/L NaHCO3 for 10 min, then boiled in 10 mmol/L EDTA for 10 min, then rinsed in deionized water, stored in 50% ethanol, and kept at 4℃ for reserve, then washed the bag in distilled water, used for dialysis of ammonium sulfate from the isolate. Ammonium sulfate.
(13) Eppendorf 5415 C or equivalent benchtop centrifuge.
(14) Silanized 1.7 ml microcentrifuge tubes.
(15) 1 moI/L MgCl2 solution.
(16) Cup-angle ultrasonic probe: 2-inch diameter, manufactured by Branson Ultrasonic (Danbury, CT, USA) or equivalent. Mg2+ precipitated SR proteins were resuspended by ultrasound in buffer D. The Mg2+ precipitated SR proteins were resuspended by ultrasound.
2. Isolation of single SR proteins from denaturing polyacrylamide gels
(1) 16 cm high vertical polyacrylamide gel electrophoresis apparatus, 0.75 mm thick spacer and comb for gel preparation.
(2) 10% acrylamide solution: acrylamide: methylenebisacrylamide 29.2:0.8. electrophoresis buffer: 0.025 mol/L Tris base, 0.192 mol/L glycine, 0.1% SDS. separator gel solution: 10% acrylamide solution, 0.375 mol/L Tris-HCl (pH 8.8 ), 0.1% SDS. concentrated gel Acrylamide solution: 4% acrylamide (acrylamide: methylenebisacrylamide 29.2:0.8), 0.125 mol/L Tris-HCl ( pH 6.8 ), 0.1% SDS.
(3) 0.25 mol/L KCl, pre-cooled at 4 °C.
(4) 2X protein spiking buffer: 0.125 mol/L Tris-HCl ( pH 6.8), 4% SDS, 20% glycerol, 10% β-mercaptoethanol.
(5) Protein elution buffer: 0.1% SDS, 50 mmol/L Tris-HCl ( pH 8.0), 5 mmol/L DTT, 0.1 mmol/L EDTA, 0.15 mol/L NaCl, 0.1 mg/ml acetylated bovine serum albumin, ready-to-use.
(6) acetone, pre-cooled to -20°C.
(7) Microcentrifuge, pre-cooled to -20°C.
(8) 6mol/L guanidine hydrochloride, prepared with Buffer D.
(9) MWCO 6000~8000 dialysis bag with microdialysis chamber. Microdialyzer: Spertrum Spcctra/por (Houston, TX, USA).
(10) Kaomas Brilliant Blue Staining Solution: 0.125% Kaomas Brilliant Blue R250 dissolved in 50% methanol and 10% acetic acid. After 20 min of staining, the color was decolorized in 50% methanol and 10% acetic acid for 15 min, then 5% methanol and 7% acetic acid.
Operation method
1. 65%~90% ammonium sulfate fraction of SR protein family was purified.
(1) In a 600 ml beaker, add appropriate amount of cells such as about 5X1011 mammalian tissue culture cells, 4X1010 insect tissue culture cells or 100 g of powdered tissue such as bovine thymus (stored at -80℃), add 330 ml of pre-cooled SR Extraction Buffer to 4℃ and place on ice. Add a stirrer to the beaker and stir the frozen powder and buffer together.
(2) Stir at 4°C for 5 min.
(3) Sonicate the cells at 4°C with a large probe, and adjust the power of the sonicator to nearly maximum. The power of the sonicator was adjusted to nearly maximum. Stirring was done every 30~60 s to ensure uniform sonication. Every 5 min, check the cell breakage with a microscope, which may take up to 15 min for tissue and only 5 min for cultured cells, and prepare a smear as a control before sonication.
(4) Transfer the samples into pre-cooled 50 ml centrifuge tubes and centrifuge the lysed cells with a JA I7 rotor at 10,000 r/min at 4°C for 20 min.
(5) After centrifugation, the tubes were placed on ice to remove the lipid layer from the upper layer of the extracts, and all the supernatants were combined in a pre-cooled 600 ml beaker and placed on ice. The exact volume of the extract was determined using a pre-cooled 500 ml measuring cylinder.
(6) Add a magnetic stirrer to the beaker and stir slowly at 4°C. Slowly add finely ground ammonium sulfate to achieve 65% saturation within 15-20 min. Ensure that the ammonium sulphate is a fine powder before use and that it can be pulverized in a mortar and pestle. (To achieve the same 65% saturation, add 0.43 g of ammonium sulphate per ml of extract.
(7) When all the ammonium sulfate has been added, stir at 4°C for 1.5 h. The extract should be stirred at 4°C for 1.5 h.
(8) Transfer the extract to a 50 ml pre-cooled centrifuge tube and centrifuge for 20 min at 10000 r/min with a JA-17 rotor.
(9) After centrifugation, place the tube on ice and remove the upper lipid layer.
(10) Pour the supernatant into a clean pre-cooled 50 ml centrifuge tube and centrifuge for 20 min at 10,000 r/min with a JA-17 rotor at 4°C.
(11) After centrifugation, place the tube on ice to remove any lipid layer that may be present in the upper layer.
(12) All supernatants were incorporated into a pre-cooled 600 ml beaker placed on ice and the exact volume of the extract was determined using a pre-cooled 500 ml measuring cylinder.
(13) Add a magnetic stirrer to the beaker and stir slowly at 4°C. Add finely ground ammonium sulfate to achieve 90% saturation within 15-20 min (to achieve 90% saturation, add 0.19 g ammonium sulfate per ml of ammonium sulfate-containing extract).
(14) Seal the mouth of the beaker with plastic film and stir the extract overnight.
(15) The next day, the samples were dispensed into pre-cooled microcentrifuge tubes.
(16) Centrifuge for 1 h at 25,000 r/min at 8°C using a SW28 rotor on a Beckman L7 ultracentrifuge.
(17) Slowly discard the supernatant from the centrifuge tube.
(18) Slowly add 3 ml of pre-cooled 4°C SR dialysis buffer containing 90% ammonium sulfate along the wall of the tube, being careful not to disturb the precipitate.
(19) Add 0.5 ml of SR Dialysis Buffer (without ammonium sulfate) to each centrifuge tube on ice.
(20) Combine all suspended precipitates into one tube. Rinse the tubes sequentially with 1 ml of SR Dialysis Buffer and incorporate the washings into the sample tubes so that the total volume of sample is about 6 ml.
(21) Wash the 15.24 to 17.78 MWCO 6000 to 8000 dialysis bag with distilled water, holding the bottom of the bag with a clamp.
(22) Transfer the resuspended precipitate into the dialysis bag and clamp the top of the bag.
(23) Dialyze with 1300 ml of SR dialysis buffer for 1 h at 4°C.
(24) Replace the buffer and dialyze for 2 h with 1300 ml of buffer.
(25) Replace with another 1300 ml of buffer and dialyze at 4°C overnight.
(26) The next day, the dialyzed solution is dispensed into 1 ml siliconized microcentrifuge tubes.
(27) The tubes are snap-frozen on dry ice.
(28) The solution can be stored at -80°C for long periods of time while it is being prepared.
2. Precipitation of SR proteins
(1) Thaw dialyzed 65% to 90% ammonium sulfate fractions in an ice bath.
(2) Remove precipitated proteins by centrifugation in a microcentrifuge at 14,000 g (13,000 r/min) at 4°C for 30 min. For larger protein samples, the dialyzed fractions can be transferred into 15 ml Corex tubes and centrifuged at 10,000 r/min (10,000 g) at 4°C for 30 min using a JA17 or equivalent rotor.
(3) Transfer the clarified supernatant to a new pre-cooled Corex tube or siliconized microcentrifuge tube using a pipette, avoiding aspiration of the precipitate and taking care that some of the supernatant is lost rather than brought in as precipitate.
(4) Add 1 mol/L MgCl2 solution to the supernatant to a final concentration of 15 mmol/L, mix well, and place the centrifuge tube on ice for 1 hour.
(5) Centrifuge the MgCl2-treated solution as in step (2).
(6) Discard the supernatant and pipette a small amount of SR dialysis buffer (containing 20 mmol/L MgCl2 ) into the centrifuge tube to wash the precipitate. Use a small amount to cover the precipitate, remove as much of the wash buffer as possible, and centrifuge briefly to ensure that the wash buffer concentrates at the bottom of the tube and is completely removed.
(7) Resuspend the precipitate in Buffer D. For precipitates in Corex tubes, aspirate 200~400 μl of Buffer D and gently blow up and down, taking care to do so gently to avoid sample loss. Transfer the resuspended precipitate to a siliconized microcentrifuge tube, rinse the tube with 100 μl of Buffer D, and incorporate the rinse solution into the microcentrifuge tube. For the precipitate in the microcentrifuge tube, add 20 to 100 μl of Buffer D per milliliter of precipitate from the 65% to 90% dialyzed ammonium sulfate fraction, taking care not to blow the precipitate up and down. In both cases, place the microcentrifuge tubes in a 4°C cup-angle sonication bath, set the power of the cup-angle probe to maximum, and sonicate for 30 s. Repeat if necessary, taking care to keep the sonication bath at 4°C.
(8) The purity of the resuspended SR proteins can be identified by SDS PAGE gel electrophoresis analysis, comparing the results of the same protein's Caulophylline blue staining with the results of immunoblotting with the mAb104 antibody. As a rule of thumb, silver staining methods are not suitable for the detection of SR proteins.
3. SDS-PAGE purification and replication of single SR proteins
(1) Prepare a 5-8 cm wide, 4 cm high 10% concentrated preparative gel with 0.75 mm thick side strips and allow the gel to polymerize for several hours before use.
(2) Add an equal amount of 2X Protein Sampling Buffer to the SR protein solution that has been precipitated by magnesium ions and resuspended in Buffer D. For the preparative gels described above, the amount of SR protein obtained from 100 g of calf thymus gives the best recovery. Care should be taken to keep the sample volume small during resuspension and not to resuspend the precipitate directly with Protein Sampling Buffer or to add Sampling Buffer to the tubes used for magnesium-ion precipitated proteins.
(3) Heat the sample at 90°C for 5 min.
(4) Sample is spiked into the prepared gel and pre-stained with MQP protein, leaving 1% of the sample to be spiked separately so that the lane is adjacent to the pre-stained MQP lane.
(5) Electrophoresis at 100 V for 7 h, or electrophoresis of the indicator to the bottom of the gel.
(6) Remove the gel, cut off the pre-stained molecular mass standard and the separate sample lane, and stain with Coomassie Brilliant Blue for record.
(7) Soak the gel containing the prepared lanes and the pre-stained molecular mass standard protein lanes in 500 ml of 0.25 mol/L KCl pre-cooled at 4℃ for 10 min.
(8) Place the gel on a glass plate and then on a black background. In the presence of proteins, KCl may interact with the SDS in the proteins to form a visible bright white precipitate.
(9) Cut off the individual protein bands separately with a razor blade and transfer to a 15 ml conical tube.
(10) Add 10 ml of 1 mmol/L DTT pre-cooled at 4°C to each tube and soak for 3 min at room temperature, then discard the solution from the tube.
(11) Place the mortar in an ice bath, add liquid nitrogen to the mortar, transfer the gel into the liquid nitrogen with tweezers, and slowly grind the gel into a fine powder.
(12) Using weighing paper pre-cooled on dry ice, transfer the powdered, crumbled gel from the mortar into a 15 ml conical tube or a siliconized 1.7 ml microcentrifuge tube.
(13) Add 0.5 to 2.0 ml of freshly prepared elution buffer, adjusting the amount of buffer according to the amount of gel, preferably so that the volumes of powdered gel and elution buffer are equal.
(14) Spin and mix overnight at room temperature to elute proteins from the gel.
(15) Centrifuge at high speed for 5 min at room temperature to separate the solution from the gel.
(16) Pipette the supernatant into siliconized microcentrifuge tubes or, if large, divide the supernatant into multiple tubes, taking care not to exceed 800 μl per tube.
(17) Centrifuge at high speed for 5 min to precipitate any gel that may be present.
(18) Transfer the supernatant to a new siliconized microcentrifuge tube, taking care not to bring up any gel that may be present, and measure the volume of supernatant in each tube with a pipette.
(19) Place the tubes in an ice-salt bath (5 mol/L NaCl solution mixed with ice) at -20 °C for 10 min.
(20) Add acetone pre-cooled to -20 °C to each tube, quickly mix the tubes upside down, and continue to place in the ice-salt bath.
(21) Place the acetone precipitate in the ice-salt bath at -20 °C for 20 min.
(22) Place the tube in a centrifuge pre-cooled to -20°C and centrifuge at 13,000 g for 20 min.
(23) Quickly remove the tube from the centrifuge and place in an ice-salt bath and immediately place in a cold room.
(24) In the cold room, discard as much supernatant as possible and add 100 μl of 6 mol/L guanidine hydrochloride in Buffer D to the tube.
(25) Place the tube at 4°C for 5 min.
(26) Place the tube at room temperature with the cap open for 15 min to allow residual acetone to evaporate.
(27) Cap the tube and sonicate for 30 s to facilitate resuspension of the precipitate.
(28) Leave the tube uncapped for 15 min at room temperature.
(29) Centrifuge at 13,000 g for 10 min at room temperature to remove protein insoluble in guanidine hydrochloride.
(30) Add the supernatant to a microdialysis device and dialyze the samples in Buffer D containing 5 mmol/L β-phosphoglycerol at a flow rate of 1 ml/min with a peristaltic pump using a MWCO 6000-8000 dialysis bag at 4°C. The samples should be dialyzed for a minimum of 3 h but not more than 7 h. The samples should be dialyzed at a flow rate of 1 ml/min with a peristaltic pump.
(31) Transfer the dialyzed solution into a microcentrifuge tube and rinse the dialysis wells with 50 μl of Buffer D. Incorporate the washings into the protein sample.
(32) Centrifuge at 13,000 g at 4°C for 30 min to remove insoluble proteins resulting from the dialysis of guanidine hydrochloride.
(33) Transfer the supernatant to a new microcentrifuge tube and add 1 mol/L MgCl2 to a final concentration of 20 mmol/L. The supernatant is then transferred to a new microcentrifuge tube.
(34) Place the tube on ice at 4°C overnight.
(35) Centrifuge at 13000 g for 30 min at 4°C.
(36) Remove the supernatant and add 20-60 μl of Buffer D to the precipitate.
(37) Set the sonicator power to maximum and sonicate for 30 s in an ice water bath to resuspend the precipitate.
(38) Take 2~4 μl of the sample for SDS-PAGE gel electrophoresis, and identify the purity of SR protein by staining with Coomassie Brilliant Blue. At the same time, a known amount of protein was doubly diluted, and the sample was applied to the same gel, and the concentration of the prepared SR protein was roughly determined according to the intensity of the staining with Caulophylline blue.