Experiments on the preparation of inhomogeneous ribonucleoprotein complexes
Experiments on the preparation of inhomogeneous ribonucleoprotein complexes
In the nucleus, the transcription products of RNA polymerase II bind to a large number of different types of nuclear precursor mRNA/mRNA-binding proteins, heterogeneous nuclear ribonucUoprotein (hnRNP), to form the hnRNP complex. This experiment is based on the "RNA Laboratory Guidebook", edited by Xiaofei Zheng.
Operation method
Experiments on the preparation of inhomogeneous ribonucleoprotein complexes
Principle
In the nucleus, the transcription products of RNA polymerase II bind to a large number of different types of nuclear precursor mRNA/mRNA-binding proteins, heterogeneous nuclear ribonucUoprotein (hnRNP), to form the hnRNP complex.
Materials and Instruments
HeLa S3 cell antibody Move I. Materials and equipment For more product details, please visit Aladdin Scientific website.
immunopurification buffer
Protein A-Sepharose gel Unidirectional gel electrophoresis Bidirectional non-equilibrium pH gel electrophoresis
1. Cellular and metabolic labeling proteins
(1) Culture of HeLa S3 cells: DMEM medium containing 10% HeLa S3 cells and 1% penicillin/streptomycin.
(2) HeLa S3 cell labeling: methionine- and cystine-free DMEM, 10% DMEM, 5% fetal calf serum ( FCS), 1% penicillin/streptomycin, and 20 μCi/ml TRANS ( ICN, COSTAMESA, CA, USA).
2. Antibody and protein A-Sepharose gels
(1) Antibody: Any monoclonal antibody with specificity and high affinity for a particular hnRNP protein can be used as an immunoaffinity purification. Monoclonal antibodies that have been successfully applied are 4B10 ( anti-hnRNP A1), 4F4 (anti-hnRNP C1/C2) and 4D11 (anti-hnRNP).
(2) Protein A-Sepharose ( pharmacyia Biotech, Piscataway, NJ. USA).
3. Immunopurification buffer
(1) Immunopurification buffer 1 ( IPB1 ): RSB-100 [ 10 mmol/L Tris-HCl (pH 7.4), 100 mmol/L NaCl, 2.5 mmol/L MgCl2 ], 0.5% Triton X-100, 1 μg/ml leupeptin, 1 μg/ml pepsin inhibitor ( peppstatin ), 0.5% ( V/V ) aprotinin.
(2) Immunopurification buffer 2 (IPB2): the composition is the same as that of IPB1 except for the absence of Triton X-100.
(3) Immunopurification buffer 3 (IPB3): PBS, 1 mmol/L EDTA, 1% dimethyl betaine (Empigen BB), 0.1 mmol/L DTT.
(4) Immunopurification buffer 4 (IPB4): PBS, 1 mmol/L EDTA, 1% Triton X-100, 0.5% deoxycholic acid, 0.1% SDS, 0.5% proteolytic peptide. IPB4 can be prepared as a 5X storage solution, and stored at 4 ℃ for no more than one month.
4. Unidirectional gel electrophoresis
(1) Reagents: 10% (m/V) SDS (autoclaved and stored at room temperature); 3% (m/V) ammonium persulfate (APS), 1 ml each, stored at -20℃; TEMED.
(2) Acrylamide storage solution for gel separation: 167.5 g (33.5%) of acrylamide and 1.5 g (0.3%) of methylene bisacrylamide were dissolved in 45 ml of water with stirring until completely dissolved, water was added to 500 ml, and the solution was filtered through a Whatman No. 1 filter paper. Store in a brown bottle at 4°C for at least 2 months.
(3) Separation gel buffer: 1 mol/L Tris-HCl (pH 9.1). 4°C for at least 3 months.
(4) Acrylamide Storage Solution for Gel Concentration: Dissolve 60 g (30%) of acrylamide and 0.88 g (0.44%) of methylene bisacrylamide in 175 ml of water with stirring until completely dissolved, replenish to 200 ml, and filter through Whaman No. 1 filter paper. Store in a brown bottle at 4°C for at least 2 months.
(5) Concentrated Gum Buffer: 0.5 mol/L Tris-HCl (pH 6.8 ). Store at 4°C for at least 3 months.
(6) 2X Sampling Buffer: 5 ml 0.5 mol/L Tris-HCl (pH 6.8), 8 ml 10% SDS, 4 ml redistilled glycerol, 2 ml β-mercaptoethanol, 2 mg bromophenol blue, replenish with water to 20 ml. Store at -20°C for at least 4 months.
(7) 4X electrophoresis buffer: 96 g Tris base, 460.8 g Glycine, 32 g SDS in water to 8 L. Store at room temperature away from light.
(8) Equipment: Vertical gel electrophoresis device.
5. bidirectional non-equilibrium pH gel electrophoresis
(1) Reagents: Ultrapure urea; 10% NP-40; 40% Bin-Lyte 3/10 amphoteric electrolyte (Bio-Rad); 10% APS; TEMEI).
(2) isofocusing Acrylamide storage solution: 28.38 g of acrylamide and 1.62 g of methylene bisacrylamide were dissolved in 90 ml of water, stirred until completely dissolved, and then replenished to 100 ml. Filtered through Whatman No. 1 filter paper, and stored in brown bottles for 3 months.
(3) Upper bath buffer (0.01 mol/L phosphoric acid): 1 ml of 85% phosphoric acid dissolved in 470 ml of water.
(4) Lower bath buffer (0.02 mol/L NaOH): 12 ml of 1 mol/L NaOH was added to 600 ml of water and degassed with a vacuum pump for 5 min.
(5) Sample buffer: 1.43 g of ultrapure urea, 0.5 ml of 10% Nonidet P-40, 0.125 ml of 40% ampholines were added to 2.93 ml with water and divided into 100 μl portions, which can be stored at -70℃ for 1 year.
(6) 95% Sample Extraction Buffer: Mix 12.7 ml 0.5 mol/L Tris-HCl (pH 6.8), 8 ml glycerol, 10 ml 0.1% bromophenol blue, 20 ml 10% SDS, replenish with water up to 95 ml and store at room temperature. Immediately before use, β-mercaptoethanol was added to a final concentration of 5%.
(7) Other devices: tubular gel electrophoresis device, vertical gel electrophoresis device, test tube rack, tuberculin syringe.
II. Methods of operation
1. Preparation of nucleoplasm
(1) Cultivate HeLa cells until most of them are fused, and then use [ 35S ] methionine for protein labeling. After pouring off the culture medium, wash the cells once with sterile PBS, add 5 ml of cell labeling medium to each 10 cm dish, and incubate them overnight (8~12 h). All the following operations were carried out in 1.5 ml centrifuge tubes at 4°C, and about half a dish of cells was needed for each affinity purification.
(2) Wash each 10 cm dish twice with 5 ml PBS and add 1.5 ml IPB1, then scrape off the cells thoroughly with a cell scraper. Blow the cell suspension four times with a 25-gauge needle attached to a syringe, avoiding foaming.
(3) Centrifuge the lysate at 3000 g for 3 min.
(4) Resuspend the XL nuclei precipitate with 0.5 ml IPB2 and sonicate the lysate 3 times in an ice bath, with 15 seconds in between.
(5) Add 0.5 ml of 30% sucrose (prepared with IPB2) to a centrifuge tube, gently spread the sonicated solution on the surface of the sucrose solution and centrifuge at 4800 g for 15 min.
(6) Remove the nucleoplasmic fraction above the sucrose solution (about 0.5 ml) and add Triton X-100 to a final concentration of 0.5%.
2. Immunopurification and gel analysis of hnRNP complexes
In this method, the hnRNP complex is purified using a monoclonal antibody against hnRNP. Immunopurification is performed under non-ionic detergent conditions to avoid disrupting protein-protein and protein-RNA interactions. The procedure should be rapid to minimize nuclease action, and the monoclonal antibody is preconjugated to Protein A Sepharose particles and incubated with the nucleoplasm for no more than 15 min.
(1) All steps are performed at 4°C. Add 1 ml of IPB1 to the pellet. Add 1 ml of IPB1 to a centrifuge tube, then add 1-5 μl of ascites, 3 μl of rabbit anti-mouse IgG, and 50 μl of Protein A-Sepharose (50%, prepared in PBS), and then place in a mixer and react for 1 h at 4℃.
(2) Wash with 1 ml of lPB1 by vortexing 3 times, centrifuge for 3 s, and discard the supernatant.
(3) Add 250 μl of labeled nucleoplasm to each tube of Protein A-Sepharose pellet containing about 25 μl of antibody-conjugated Protein A. Mix at 4℃ for 15 min.
(4) The Sepharose gel pellet was washed five times with IPB1, 1 ml each time, and then washed once with 1 ml RSB-100.
(5) Remove excess liquid with a syringe equipped with a 27-gauge needle, but do not allow Protein A-Sepharose to dry too long.
(6) Analyze by unidirectional gel electrophoresis, add 40 μl of 2X electrophoresis sample buffer, heat at 100°C for 3 min, centrifuge at maximum speed for 2 min on a microcentrifuge, and sample 20 μl of each lane.
(7) Analyze by two-way gel electrophoresis, add 20 μl of the same buffer and 2 μl of 20 mmol/L DTT, mix and incubate at room temperature for 5 min, centrifuge for 30 s, and centrifuge the supernatant for another 30 s. Sample 18-20 μl of each lane according to the procedure (1) to (5) of operation 5 "two-way gel electrophoresis". Electrophoresis was performed according to operation 5 "Bidirectional gel electrophoresis" steps (1)~(5).
2. Immunopurification and Gel Analysis of Specific hnRNP Proteins
Immunoaffinity purification of proteins that are only specifically recognized by monoclonal antibodies (especially if the protein is not a known component of the hnRNP complex) is very useful and can be carried out by either direct immunopurification from labeled cell extracts or purification from purified hnRNP complexes, as described below. Each is described below:
(1) Purification from labeled cell extracts. The previous steps are the same as steps (1) and (2) in operation 1, "Preparation of nucleoplasm", except that the nuclei are precipitated by resuspension of the cells with IPB3 or IPB1 instead of IPB1, followed by direct sonication of cell lysates [step (4) in operation 1, "Preparation of nucleoplasm"]. ]. Remove the precipitate by centrifugation in a centrifuge tube at maximal speed for 10 min, and then perform immunopurification as described in operation 2, "Immunopurification and gel analysis of hnRNP complexes" (using IPB3 or IPB4 instead of IPB1 ).
(2) Immunopurification from immunopurified hnRNP. Purify the hnRNP complex according to steps (1) to (5) of operation 1 "Immunopurification and gel analysis of hnRNP complex". Add 50~100 μl of RSB-100 containing 1% SDS and heat at 100℃ for 3 min to dissociate the complex, then cool to room temperature and dilute 10-fold with IPB4 (to reduce the concentration of SDS to about 0.1%). Finally, the protein was purified (IPB4 instead of IPB1) as described in Procedure 2, "Immunopurification and Gel Analysis of hnRNP Complexes".
4. Unidirectional gel electrophoresis
The discontinuous buffered gel system used in this experiment has good resolution for both low molecular mass halo and high molecular mass hnRNP and is not easily ruptured when dried in a vacuum gel dryer.
(1) Preparation of 12.5% separator gel. Mix 14.8 ml separation gel with acrylamide storage solution, 15.2 ml electrophoresis buffer, 8.3 ml water, 0.4 ml 10% SDS, 1 ml 3% ammonium persulfate (APS), and 20 μ TEMED. The gel was perfused in an electrophoresis apparatus and the upper layer was covered with 0.1% SDS and left to polymerize.
(2) Preparation of concentrated gel. Mix 1.3 ml of concentrated gel with acrylamide storage solution, 2.5 ml of 0.5 mol/L Tris-HCl ( pH 6.8), 6.1 ml of water, 0.1 ml of 10% SDS, 0.1 ml of ammonium persulfate (APS ) and 25 μl of TEMED. The polymerized separation gel was filled with approximately 1 cm high concentrated gel and the comb was inserted.
(3) Using a Hoefcr SE400 gel electrophoresis setup, 20 μl of the protein sample was first concentrated by electrophoresis at 80 volts, and then electrophoresed at 150 V for approximately 5 h until the bromophenol blue was completely swept out of the gel.
(4) Fixation and staining. The gel was fixed and stained in 10% acetic acid, 25% isopropanol and 0.025% (m/V) komassie blue overnight, and then decolorized with 10% acetic acid for 2 h. The gel was then stained with 10% acetic acid for 2 h, and then decolorized with 10% acetic acid.
(5) Development. The gel was soaked in DMSO for 15 min, poured off the DMSO and added new DMSO for another 15 min. DMSO was poured off, and DMSO containing 22% 2,5-diphenyloxazole (PPO) was soaked for 1 h, then poured off and washed with running water for 1 h. The gel was dried in a heated vacuum gel dryer for 1.5 h. The gel was then dried in a heated vacuum gel dryer for 1.5 h. The gel was then washed with water and washed with water.
5. Bidirectional gel electrophoresis
Since the hnRNP complex consists of a large number of proteins, bi-directional electrophoresis can better reflect the complexity of these complexes. Since the human hnRNP complex contains a large number of basic proteins, unbalanced pH gel electrophoresis was used for the first direction electrophoresis.
(1) Preparation of the first direction gel. In a 10 ml beaker with a stirrer, add 1.38 g of ultrapure urea, 0.5 ml of 10% NP40, 125 μl of 40% amphoteric electrolyte, 0.49 ml of water, and 0.33 ml of isoelectrically focusing acrylamide reservoir, which is enough to make 9 tubes of gel. Cover the beaker with a sealing film and stir and mix for 30 min until the urea is completely dissolved. Do not heat the acrylamide solution.
(2) While the acrylamide solution is being mixed, spread the modeling clay on a clean bench and cover with masking tape. Wrap two rubber bands around the periphery of the test tube holder and press the holder into the clay while adding a 200 μl capillary tube to the front of the tuberculin syringe (mark 11.5 cm from the bottom).
(3) After preparing the gel solution, 4 μl of 10% APS and 5 μl of TEMED were added, mixed well, and the acrylamide solution was aspirated with a syringe to a level just above the 11.5 cm mark. The capillary tube was then passed through a rubber band secured to the periphery of the test tube holder and fixed to the clay at the bottom, the liquid level was adjusted to just past the 5 cm mark and checked for air bubbles.
(4) After all gels were filled, the top of each gel was covered with 15 μl of 8 mol/L urea and covered with a sealing film. Polymerize for 4 h.
(5) Gel. After polymerization, remove the covering urea. Select gels of equal length.
(6) Add degassed NaOH solution to the lower tank of the tubular gel unit.
(7) Use a syringe with a 21-gauge bent needle to suck out any air bubbles at the bottom of the tube.
(8) Insert the hose into a tube rack with rubber fittings, place the rack in the lower tank, and fill the upper tank with 0.01 mol/L phosphoric acid solution.
(9) Before loading the sample, rinse the tube with the upper tank solution using an extended pipette tip. Sample, invert the electrode and electrophoresis at 400 V for 4 h.
(10) After electrophoresis, slowly squeeze the gel into a 15 ml conical flask containing 4.75 ml of sample buffer using a 20 ml syringe.
(11) Add 250 μl of mercaptoethanol to each tube, mix gently, equilibrate at room temperature for 5 min, freeze rapidly in a dry ice/ethanol bath, and store at -80 °C.
(12) Preparation of second-way gel. Perfuse the gel with a spacer of 1.5 mm thickness as described in steps (1) to (3) in operation 4 "Unidirectional gel electrophoresis", and insert a single-hole comb about 0.5 cm into the concentrated gel.
(13) Rapidly thaw the gel in the first direction in a 37℃ water bath with gentle stirring.
(14) Carefully place the gel in the position of the comb and straighten the gel with flat-tipped forceps. Determine the position of the gel between the glass plates and gently push the first-way gel on top of the gel concentrate.
(15) Perform second-way electrophoresis until the bromophenol blue runs out of the gel. Then fix, stain, and develop [ see steps ( 4)~(5) in 4 "Unidirectional Gel Electrophoresis" ].