How to Select a Western Blotting Film in a Western Blotting Experiment

Nitrocellulose membrane (NC membrane) is the most commonly used blotting membrane for protein and nucleic acid hybridization and is the standard solid support for Western blotting experiments. In a low ion transfer buffer environment, most negatively charged proteins bind to the nitrocellulose membrane with hydrophobic interactions and bind with high affinity, although the mechanism is not yet clear, but due to the nitrocellulose membrane This feature, but also easy to block non-specific binding, which has been widely used. There are many NC film products on the market today and their quality is uneven.

From the texture of the membrane, the most important indicator is the amount of protein that can be bound per unit area. The binding ability of the nitrocellulose membrane is mainly related to the purity of the nitrocellulose membrane. Some nitrocellulose membranes on the market usually have a large amount of cellulose acetate, which reduces the amount of protein bound. If 100% pure nitrocellulose is used, maximum protein binding is guaranteed, up to 80-150 μg/cm2. Sartorius's NC membranes are dense 100% nitrocellulose membranes that are excellent filters for Western, Northern and Southern hybridizations. Due to 100% purity, it also greatly reduces the non-specific binding, reduces the hybridization background, and ensures maximum protein binding, up to 150-200 μg/cm2. In addition, Sartorius's superior NC film has good toughness and strength. Compared with the traditional NC film, the texture is relatively brittle, and it is broken once or twice. The advantage of Sartorius's NC film is obvious. It can be used repeatedly and is not easily damaged. It is the best choice for Western, Northern and Southern hybridization experiments. .

From the point of view of membrane selection, different pore sizes of nitrocellulose membranes are selected depending on the size of the protein to be transferred. Because with the continuous decrease of the pore size of the membrane, the stronger the binding of the membrane to the low molecular weight protein is. However, if the membrane pore size is less than 0.1 mm, protein transfer is difficult. Therefore, we usually use two kinds of nitrocellulose membranes of 0.45μm and 0.2μm. For a protein larger than 20 kD, a 0.45 μm membrane can be used. For a protein smaller than 20 kD, a 0.2 μm membrane should be used. If a 0.45 μm membrane is used, a "Blow through" phenomenon occurs. Sartorius's NC membranes are also available in two options that can cover almost any user's needs. Nitrate membranes with a pore size of 0.2 μm are recommended for the transfer of small proteins and nucleic acids, which can avoid sample loss due to membrane transfer; nitrocellulose membranes with a pore size of 0.45 μm are recommended for the transfer of macromolecular proteins and nucleic acids. As well as most analytical blots, including protein ssDNA and RNA transfer.

From the point of view of the use of the film, the NC film on the market needs to be pretreated before use, and there is also no need for the pretreatment to be used directly. Sartorius's NC membrane does not require pretreatment with anhydrous methanol. The membrane only needs to be wetted in water and then placed in the transfer buffer. Simple operation can be used with confidence.

Therefore, Sartorius's NC membrane is an ideal choice for related hybridization research experiments.

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