Gel blocking assay to analyze RNA-protein interaction constant assay
The simplicity of the gel reiardadon assay (also known as mobthty shift assay) method, its sensitivity, and its ability to be quantified have made it widely used in the study of transcription and gene regulation. It is now commonly used to purify RNA (or DNA) binding proteins, to determine the sequence recognized by a known or possible RNA binding protein, to establish reaction constants (e.g., affinity constants and dissociation constants), and so on. In this lab source "RNA Lab Guidebook" edited by Xiaofei Zheng.
Operation method
Gel blocking experiments to analyze RNA-protein interaction constant experiments
Principle
The simplicity of the gel reiardadon assay (also known as mobthty shift assay) method, its sensitivity, and its ability to be quantified have made it widely used in the study of transcription and gene regulation. Currently, it is commonly used to purify RNA (or DNA) binding proteins, to determine the sequence recognized by a known or possible RNA binding protein, to establish reaction constants (e.g., affinity constants and dissociation constants), and so on.
Materials and Instruments
Protein RNA to be analyzed Move I. Materials and equipment For more product details, please visit Aladdin Scientific website.
Binding buffer Electrophoresis storage solution
Electrophoresis apparatus Electrophoresis tanks Glass plates Gaskets Combs Polyacrylamide gel electrophoresis X-rays Dark boxes Radiological dosimeters
1. labeled RNA fragments, unlabeled homologous RNA.
2. proteins to be analyzed.
3. DEPC-treated water.
4. 10X binding buffer: 200 mmol/L Tris-HCl (pH 7.5), 500 mmol/L KCl, 50 mmol/L MgCl2, 10 mmol/L dithiothreitol, 10% glycerol, 1 mg/ml large-size bovine albumin. 2X storage solution of this buffer is also available for optional use.
5. Polyacrylamide gel electrophoresis was performed using a standard electrophoresis apparatus, electrophoresis tanks, glass plates, spacers and combs.
6. electrophoresis storage solution and reagents: 40% acrylamide, 2% methylene bisacrylamide, 80% glycerol, 4X Tris Glycerol buffer (1X buffer is 25 mmol/L Tris base, 0.2 mol/L glycerol), ammonium persulfate, TEMED.
7. x-ray film, dark box, appropriate dosimeter for radioactive substances.
II. Methods of operation
1. RNA and protein binding reaction
(1) In an ice bath, add the following reagents to a microcentrifuge tube and mix well: 1 ng of end-labeled RNA fragments, 2 μl of 10X binding buffer, appropriate amount of end-labeled homologous RNA, appropriate amount of protein to be tested, and add water to 20 μl.
(2) Incubate the reaction solution at 37°C for 30 min. The temperature and time should be determined empirically, but for most reactions, 30 min is sufficient to reach equilibrium.
2. Non-denaturing polyacrylamide gel electrophoresis
(1) The gel can be prepared in advance. Thoroughly clean the glass plate with water and ethanol, using a 0.7 mm thin spacer, a 12-tooth comb with each tooth measuring 0.8 cm X 1.5 cm, and a glass plate measuring 18 cm X 23 cm.
(2) Mix the appropriate storage solution to obtain a 40 ml solution containing 6% acrylamide, 0.12% methylene bisacrylamide, 25 mmol/L Tris base, 0.2 mol/L glycine, 5% glycerol, add 15 mg of ammonium persulfate and TEMED, perfuse between the two glass plates, insert the comb at a depth of 1~1.2 cm, and wait until the polymerization is complete, and then equilibrate the gel at the final electrophoresis temperature. temperature to equilibrate the gel.
(3) Remove the bottom spacer, load the gel into the electrophoresis tank, add electrophoresis buffer (25 mmol/L Tris base and 0.2 mol/L glycine) to the top and bottom tanks, blow every well with a pipette to make sure that any air bubbles at the bottom of the gel are removed from the wells, and pre-electrophoreze the gel at 300V for not less than 30 min before sampling.
(4) Sample, electrophoresis at 300 V for 3 h.
(5) Empty the electrophoresis tank and remove the gel. All radioactivity, including unbound nucleoside triphosphates from the RNA labeling reaction, remains in the gel, and the buffer in the bottom tank should be free of radioactivity, but any change in the test parameters must be determined by determining whether or not there is any radioactivity in the buffer in the bottom tank (which is necessary in all cases). Remove a glass plate and, depending on the method of quantification, the gel may be dried on Whatman filter paper in a gel dryer, fixed (12% methanol, 10% acetic acid), or quantified directly by wrapping the wet gel in plastic wrap.
3. Quantification of results
The amount of free RNA and bound RNA radioactivity corresponding to each band can be determined in several ways:
(1) Radiographic autoradiography and optical densitometry. Wet or dry gels can be X-ray developed, and the results of radiographic autoradiography can be analyzed by optical density scanning. The biggest disadvantage of this method is that the inherent low linear range of response of X-ray quantification can lead to large quantitative errors, and if this method is used, it is necessary to establish a standard curve to convert the units of optical density to the units of measurement of the radioactivity to reduce the errors caused by the nonlinear response of the X-rays. The method must be used to convert the optical density units to the radiographic units of measurement to minimize the errors associated with the nonlinear response of the X-ray film.
(2) Wet gels can be exposed directly to X-rays, followed by localization of the bands on the gel, which can be cut off and counted on a liquid scintillation counter. Although this method avoids the problem of X-ray film optical density nonlinearity, it is cumbersome and prone to errors in cutting the gel.
(3) Photographic imaging analysis technique. The dried gel can be exposed to a phosphor screen and then processed and analyzed using a commercial photographic imager and corresponding analysis software, which is accurate and superior to the methods described previously.
(4) Direct Detection of β Particle Emission. Instruments for the direct detection of β-particle emission from gels can be purchased from companies (Amibis/Scanalytic, BetaScope, and Packard) that typically do not have as high a resolution as photographic imaging techniques, but are adequate for analyzing data from gel-blocking assays, and usually require only a short scanning time for the quantification of labeled RNA on the gel. Quantification of labeled RNA on gels is usually accomplished with a short scanning time.
Regardless of which method is used for quantification, the final result must be representative of the amount of radioactivity in the bound or free RNA, and can be in any unit as long as it is appropriate for the test. Separation of RNA-protein complexes may occur during electrophoresis, resulting in dispersion of radioactivity in front of low mobility complexes, which should be counted as part of the RNA-protein complex.