Experimental purification and removal of nucleoprotein complexes by antisense affinity chromatography
Experimental purification and removal of nucleoprotein complexes by antisense affinity chromatography
This experiment was derived from "RNA Laboratory Guidebook", edited by Xiaofei Zheng.
Operation method
Experimental purification and removal of nucleoprotein complexes by antisense affinity chromatography
Materials and Instruments
HeLa cells Move I. Materials and equipment For more product details, please visit Aladdin Scientific website.
NaCl Wash Laemmli Sampling Buffer ATP Creatine Phosphate Sterile Distilled Water Urea Buffer MD 0.1 MD 0.6 KCl Wash Streptavidin Agarose Gel Closure Buffer NP-40
Streptavidin Agarose Gel Dounce Glass Homogenizer Micro Dialysis Cups Dialysis Bags
1. AAS method for RNP purification
The buffer should be freshly prepared and pre-cooled to 4°C. Pefaloc (Boehringer) and DTT should be added to the buffer before use.
(1) Biotinylated 2'-O-methyl RNA oligonucleotides can be synthesized on a Pu Bu DNA Synthesizer. 2'-O-allyl RNA oligonucleotides can also be used instead of 2'-O-methyl RNA oligonucleotides in experiments, which reduces its binding to non-specific proteins.
(2) Streptavidin agarose gel.
(3) Nucleus extracts are preferably prepared from freshly cultured Hela cells or directly from purchased frozen cells.
(4) Dounce glass homogenizer, pestle and mortar type A and type B.
(5) 1 ml microdialysis cup (12000 Da molecular mass cutoff).
(6) 100 mmol/L NaCl wash solution (WB 100): 0.1 mol/L NaCl, 20 mmol/L HEPES (pH 7.9), 0.05% NP 40.5 mmol/L Pefablock, 0.5 mmol/L DTT.
(7) 250 mmol/L NaCl wash (WB 250): change the NaCl concentration in WB 100 to 250 mmol/L.
(8) Laemmli Sampling Buffer: 10 ml buffer containing 2.4 ml 1 mol/L Tris-HCl (pH 6.8), 3 ml 20% SDS, 3 ml glycerol, 1.6 ml β-mercaptoethanol, and 0.006 g bromophenol blue.
(9) 1 mol/L MgCl2.
(10) 1 ml ATP (pH 7.0).
(11) 0.5 mol/L phosphocreatine (pH 7.0).
(12) 3 L of pre-cooled sterile distilled water.
(13) 8 mol/L urea.
(14) 1 mol/L NaCl (WB 1000): the concentration of NaCl in WB 100 was changed to 1 mol/L.
2. Removal of RNP by the AAD method
The buffer was freshly prepared and pre-cooled to 4°C. Phenylmethylsulfonyl fluoride (PMSF) and DTT were added to the buffer before use.
(1) Dialysis bags with widths of 10 mm and 25 mm (retained molecular mass of 12000-14000 Da).
(2) HeLa cells and Dounce homogenizer [same as 1 "AAS method for RNP purification"].
(3) Buffer A: 10 mmol/L HEPES (pH 7.9), 1.5 mmol/L MgCl2, 10 mmol/L KCl, and 1.5 mmol/L DTT.
(4) Buffer S: 20 mmol/L HEPES (pH 7.9), 10% glycerol, 0.42 mol/L KCl, 1.5 mmol/L MgCl2, 0.2 mmol/L EDTA, 0.5 mmol/L DTT and 0.5 mmol/L PMSF.
(5) Buffer D: 20 mmol/L HEPES ( pH 7.9), 20% glycerol, 0.1 mol/L KCl, 0.2 mmol/L EDTA, 0.5 mmol/L DTT, and 0.5 mmol/L PMSF.
(6) MD 0.1: Buffer D containing 0.1 mol/L KCl and 10% glycerol.
(7) MD 0.6: Buffer D containing 0.6 mol/L KCl and 10% glycerol.
(8) 250 mmol/L KCl Wash (WB 250): 20 mmol/L HEPES (pH 7.9), 0.01% NP40, 0.5 mmol/L DTT, 250 mmol/L KCl.
(9) 0.1 mol/L ATP ( pH 7.0).
(10) 0.5 mol/L phosphocreatine ( pH 7.0 ).
(11) Streptavidin agarose gel blocking buffer: 100 mg/ml glycogen, 1 mg/ml bovine serum albumin, and 100 mg/ml tRNA (final concentration) were added to WB250 and streptavidin agarose gel.
(12) 5% NP-40, prepared with water.
II. Methods of operation
1. Purification of snRNP
(1) Preparation of pre-adsorbed cytosolic extracts
Streptavidin agarose gel has a strong property of non-specific binding of proteins and it is necessary to pre-treat it in order to avoid non-specific adsorption of proteins. Generally at least 5 ml of cell nucleus extracts are taken in 15 ml Falcon tubes for pretreatment. Unless otherwise indicated, all procedures are performed at 4°C. Streptavidin agarose gels should be centrifuged at 1300 g. Excessive rotation speeds will destroy the agarose gel beads.
① Pre-wash: Mix the streptavidin agarose gel with an equal volume of WB250, centrifuge and discard the wash solution.
② Add an equal volume of freshly prepared WB250 and divide equally into 5 portions, each agarose gel should be not less than 0.6 times the volume of the cell nuclear extract used. I.e., if 5 ml of nuclear extract is used, the streptavidin agarose gel should be at least 3 ml per portion. centrifuge and discard all washings.
③ Dilute the nuclear extract 1:1 with WB100 and add to one portion of the agarose gel.
④ Place on a rotating wheel and slowly rotate for 1 h.
⑤ Centrifuge at 1300 g for 2 min.
⑤ Centrifuge at 1300 g for 2 min. ⑥ Take the supernatant and add it to a new streptavidin agarose gel.
⑦ Repeat steps ④ to ⑥ three times.
⑧ Centrifuge twice to ensure complete removal of the streptavidin agarose gel.
⑨ If not proceeding to the next step, divide the pre-adsorbed nuclei extract into small portions, e.g., 1 ml each, flash freeze in liquid nitrogen and store at -80°C.
(2) One-step antisense affinity selection method for purification of snRNP ( AAS )
① Thaw the desired amount of pre-adsorbed nucleus extract at 30 ℃, and proceed as follows with 1 ml of pre-adsorbed nucleus extract.
② Mix the following reagents in a 1.5 ml microcentrifuge tube: 1~5 nmnol of biotinylated antisense 2'-O-methyl RNA oligonucleotide, 15 μl of 0.1 mol/L ATP, 10 μl of 0.5 mol/L phosphocreatine, 2 μl of 1 moI/L MgCl2, and add water to 70 μl.
(iii) Add 70 μl of premixed solution to 0.9 ml of pre-adsorbed cell nucleus extract and mix gently.
④ Add 30 μl of 5 mol/L NaCl so that the final concentration of NaCl in the mixture is 250 mmol/L.
⑤ Incubate at 30℃ for 1 hour.
⑥ Centrifuge at 13000 g for 30 s to remove the non-specific precipitate.
⑦ Transfer the supernatant into a new tube with 0.15 ml of streptavidin agarose gel, which should be pre-washed twice with WB250.
⑧ Mix at 4℃ for 90 min on a rotor.
Centrifuge at 1300 g.
Discard the supernatant and resuspend the streptavidin agarose gel with 0.9 ml WB250 and mix for 5 min.
(11) Repeat steps ⑨ and ⑩ three times. Remove 1/10 volume of the agarose gel slurry (approximately 0.115 ml) and store for analysis of the co-selected RNA, which is necessary to determine the efficiency and specificity of the selection.
(12) Centrifuge the residue and discard the supernatant.
(13) Resuspend with 0.8 ml 8 mol/L urea and mix by rotation at room temperature for 30 min.
(14) Centrifuge at 9000 g and transfer the supernatant to a new tube.
(15) Repeat step (14) to ensure complete removal of the streptavidin agarose gel.
(16) Remove urea by dialysis with 3 L of distilled water in a 1 ml dialysis bag.
(17) Concentrate the sample using a rotary vacuum concentrator.
(18) Resuspend proteins with Laemmli Sampling Buffer, analyze by SDS-PAGE, and detect proteins by silver staining.
(3) Regeneration of streptavidin agarose gel
① Resuspend the used streptavidin agarose gel with at least 5 times the volume of 8 mol/L urea.
① Resuspend the used streptavidin agarose gel with at least 5 times the volume of 8 mol/L urea.
③ Centrifuge and remove the supernatant.
④ Add several times the volume of WB100.
⑤ Spin and mix for 10 min, centrifuge and remove supernatant.
⑥ Repeat steps ④ to ⑤ three times or more.
(vii) Resuspend the streptavidin agarose gel with WB100 containing 0.01% NaN4 and store at 4℃.
⑧ Add 1/10 volume of fresh streptavidin agarose gel before use.
2. snRNP removal
(1) Preparation of High Salt Cell Nucleus Extracts
All of the following operations should be performed at 4°C unless otherwise indicated.
① Collect early to mid-log phase HeLa cells. Frozen cells can also be purchased and must be thawed at 30°C before use.
② Wash the cells with 5 times the cell volume of Buffer A. Collect the cells by centrifugation at 750 g for 10 min at 4℃. Repeat the wash once more.
③ Resuspend the cells with 2 times the volume of Buffer A, transfer to a 40 ml Dounce homogenizer, and crush the cells by grinding 10 times with a mortar and pestle type A. The cells can be viewed on an inverted microscope. Cell lysis can be observed on an inverted microscope. Multiple grindings can release all the inoculated nuclei, and generally 20-30 grindings are required.
④ Transfer the lysed cell suspension to a 50 ml Oak Ridge centrifuge tube and centrifuge at 450 g for 5 min at 4°C to precipitate the nuclei.
⑤ Discard the supernatant and centrifuge carefully to avoid damaging the nuclei. 25100 g for another 20 min.
(6) Discard the supernatant and resuspend the precipitate with buffer S. Use 4.5 ml of Buffer S per 109 cells.
(vii) Transfer the resuspended nuclei to a 40 ml Dounce homogenizer. Grind 10 times with a Type B mortar and pestle to observe nuclei lysis under an inverted microscope. Keep grinding until no intact nuclei are visible, usually 20-30 times. Transfer the lysate to a 15-ml Falcon tube and mix by slow rotation at 4°C for 30 min.
⑧ Centrifuge in a 50 ml Oak Ridge tube at 25100 g for 30 min at 4°C. Transfer the supernatant to a 25 mm wide dialysis bag.
Dialyze with 3 L (1 L at a time) of MD 0.1 buffer for 3.5 h. A considerable amount of precipitate will precipitate.
⑩ Centrifuge in 15 ml Falcon tubes in a bench-top centrifuge at 600 g for 10 min and discard the precipitate.
(11) Dialyze with 3 L (1 L each) of MD 0.6 buffer for 1.5 h. High salt cell nuclear extract (HSNE) was obtained.
(12) The high salt nuclear extract (HSNE) was divided into 1 ml portions, snap-frozen in liquid nitrogen and stored at -80 ℃.
2. antisense affinity removal of snRNP ( AAD )
① If using frozen high salt nuclear extract, first thaw in a 30℃ water bath.
② Add the following components to the nucleus extract: appropriate amount of biotinylated antisense oligonucleotide, 1.5 mmol/L ATP, 5 mmol/L phosphocreatine, and 0.05% NP40. If a conventional dialysis bag is used, at least 1.5 ml of nucleus extract is required, otherwise successive dilutions will result in a loss of protein activity. If only a small amount of high salt nuclear extract is available, a 1 ml microdialysis bag can be used. Generally, 2-5 ml of HSCE is used.
To avoid dilution, the volume of the substances added above should not add up to more than 10% of the total volume of the extract. That is, 16 μl of 0.1 mol/L ATP, 11 μl of 0.5 mol/L phosphocreatine, 10 μl of 5% NP40, and 40-50 μl of antisense oligonucleotide per ml of high-salt cell nuclear extract. The amount of antisense oligonucleotide depends on the amount of snRNP to be removed.
③ Incubate at 30°C for 30 min (if U1 and U2 snRNP are removed) or 1 h (if U4/U6 and U5 snRNP are removed).
④ Prepare a streptavidin agarose gel prior to incubation. The streptavidin agarose gel should be pre-contained in a containment buffer that is approximately 30% of the volume of the streptavidin agarose gel to seal off the non-specific binding site. 2 ml of extract requires 0.5 ml of streptavidin agarose gel for each cycle of the removal reaction.
⑤ Remove the pre-containment buffer by centrifugation at 1300 g for 1 min on a benchtop centrifuge and wash with 3 times the volume of new WB250 mix, repeating the wash more than twice. Divide the streptavidin agarose gel into portions according to the amount used each time.
(vi) Centrifuge at 1300 g for 1 min and twice to completely remove the WB250, which is important to avoid dilution of the extract.
⑦ Add the warmed cytosolic extract to the streptavidin agarose gel. In the case of 2 ml of extract, this can be done by splitting the extract into two equal portions, i.e. 1 ml of extract in 0.5 ml of streptavidin agarose gel in a 2 ml Eppendorf tube. The extracts can be recombined before final dialysis.
⑧ Incubate the nucleus extracts with the streptavidin agarose gel for 45 min at 0°C. It is important that the Eppendorf tubes, sealed with a sealing film, are placed in 50 ml Falcon tubes, filled with crushed ice, and spun at 4°C in a greenhouse. It is important to note that the mixture of 0.6 mol/L KCl and streptavidin agarose gel adversely affects the cytosolic extracts, so this step should be done quickly.
Remove the streptavidin agarose gel by centrifugation at 1300 g for 1 min and repeat steps 8 and 9 with a new streptavidin agarose gel.
Centrifuge twice at ⑩ 1300 g (1 min each) to remove the streptavidin agarose gel and combine the extracts.
(11) Dialyze the extracts for 1.5 to 2 h in buffer D (1 L for 3 L) using a 10 mm wide dialysis bag.
(12) Divide the extracts after snRNP removal into small portions (e.g., 50 μl each), flash-freeze in liquid nitrogen, and store at -80 °C.