The binding of proteins to DNA and RNA can be determined using nitrocellulose membrane binding, which is based on the fact that most proteins bind to nitrocellulose membranes, and if a protein binds to a nucleic acid, then the complex can also bind to the nitrocellulose membrane, but the binding must be able to withstand the filtration pressure and the protein must be able to maintain its binding to the nucleic acid while binding to the nitrocellulose membrane. Binding of the protein to the nucleic acid. The source for this experiment is the RNA Laboratory Guidebook, edited by Xiaofei Zheng.
Operation method
Determination of RNA-protein dissociation constants by nitrocellulose filter membrane binding experiments
Principle
Binding of proteins to DNA and RNA can be determined using nitrocellulose membrane binding. The method is based on the fact that most proteins bind to nitrocellulose membranes, and if a protein binds to a nucleic acid, then the complex will also bind to the nitrocellulose membrane, but the binding must be able to withstand the filtration pressure, and the protein must be able to maintain its binding to the nucleic acid as it binds to the nitrocellulose membrane. However, some proteins are denatured when bound to a nitrocellulose membrane.
Materials and Instruments
Nuclease Move I. Materials and equipment For more product details, please visit Aladdin Scientific website.
Buffers
Nitrocellulose Filter Membrane Sterilized Vials Polypropylene Microtiter Plates Spot Hybridization Devices Pipettes Microporous Glass Filtration Devices
All buffers must be prepared with deionized water that removes ions, organics, and biological contaminants, but nuclease contamination is still a common problem, and each solution must be filtered using a nitrocellulose filter membrane to remove the nuclease, with the solution stored in sterilized bottles. Use pipettes or plastic tips to transfer liquids, and use high purity reagents to avoid contamination of the nucleic acid solution, which may cause a change in the dissociation constant.
1. The apparatus used for point hybridization is as follows:
(1) 4 in. X 5 in. Schleicher and Schuell (Keene Corporation, NH, USA) 0.2 μm cellulose nitrate filter membrane ( BAS) or 4 in. X 5 in. DEAE membrane and 4 in. X 5 in. Schleicher and Schuell 0.15 μm cellulose nitrate filter membrane (BA45).
(2) 96-well polypropylene microtitration plate.
(3) Dot hybridization device.
(4) Eight-channel pipette.
2. Individual filter membrane devices:
(1) Cellulose nitrate filter membrane: Schleicher and Schuell BA85 0.45 μm or 0.2 μm.
(2) 25 mm microporous glass filters.
Store nitrocellulose filter membranes in a plastic bag at 4°C to prevent them from drying out. Older membranes are prone to uneven wetting and become fragile and unreliable when dry. It must be remembered that nitrocellulose can break when overheated or knocked, and that old ones are even more fragile.
Polypropylene microtitre plates are soaked in 1 mol/L HCl for 30 min prior to use to remove traces of RNase or adherent proteins, and can be used multiple times after this treatment. Polycarboxylate or polystyrene plates do not have hang acid properties and are more susceptible to protein adhesion on the surface.
II. Methods of operation
1. Pre-wet the nitrocellulose filter membrane at the appropriate buffer and temperature, this process should be at least 30 minutes, to ensure that all surfaces are in contact with the buffer, the membrane is not sufficiently saturated with buffer must be discarded, to avoid finger contact with the membrane.
2. Add a volume of sample to a microtitration plate, titrate increasing amounts of protein to quantitative 32P-labeled RNA, and use RNA-only wells as a control to measure the nonspecific RNA binding capacity of the membrane. A routine curve should cover 3 to 4 orders of magnitude of protein concentration, with a minimum of 5 points per group (more points are recommended to more accurately determine intermediate points), with points above (saturated) or below (unsaturated) the baseline. （The points above (saturated) or below (unsaturated) the baseline should be appropriate. Each experiment is performed twice in parallel and averaged.
3. After wetting, remove excess buffer and prepare a filter membrane by lightly sealing it with Kimwipe to prevent diffusion of the sample across the membrane.
4. Before filtration, insert the membrane into the device and turn on the vacuum pump to transfer the sample from the plate to the filter, start titrating from the lowest concentration of protein sample and gradually go up to a higher concentration, the same pipette tip can be used in one titration, too long a pipette filtration will lead to excessive retention, so this step must be done quickly, so use the same pipette tip in one titration. Air bubbles may form between the membrane and the solution and prevent filtration. These bubbles must be removed, which can be accomplished by tapping the edge of the device. Over-concentrated proteins may also bubble when aspirated, which is a sign of protein denaturation, and results will be inaccurate.
5. Remove the membrane and carefully blot up the excess buffer underneath to prevent spreading on the surface and causing individual spots to become blurred.
6. Determine the RNA bound to the filter membrane using a photographic imager, either when the membrane is dry or wet, or, if the membrane is wet, wrap it in Saran film and measure it. The amount of radiolabeled RNA bound is measured directly, and the amount bound (B) and the total amount bound (T) are compared directly. RNA bound to the membrane in the absence of protein serves as the background ( O ), which is removed from each data point to give (B-O) /T=FB (fragment binding), and the binding isotherm is obtained by plotting FB against the logarithm of the protein concentration.
7. Analyze the results of the binding assay. Complex formation is assumed to be the binding of two molecules, and the chemical formula for the binding is 1:1. The dissociation constant is obtained by looking up the value corresponding to the data in the Langmuir isotherm, and the following formula is given by Lin and Riggs: 
The constant C is appropriate above the baseline (i.e., when the retention rate is insufficient) and can be used to normalize the changing retention rate. Only after an individual data column has been normalized can it be analyzed with replicated data columns, so obtain a value for C from the initial filtering, divide each FB by the parameter C to obtain FBnom, which is used as a function of protein concentration, replace FB with FBnom, remove C (or set to 1) and do it again. To analyze individual columns of data, a Macintosh plotting software such as KaleidaGraphTM or PC is sufficient (Synergy Software), while other software packages can handle more complex analyses, including ScientistTM (MicroMath Sciennfic Software).