I am doing EMSA for antibody-DNA interaction. what should be the composition of binding buffer. can anybody help.?
The composition of the binding buffer for an EMSA for antibody-DNA interaction will vary depending on the specific antibody and DNA being used. However, a general composition that can be used as a starting point is as follows:
10 mM Tris-HCl, pH 7.5
100 mM KCl
1 mM EDTA
0.1 mM DTT
5% v/v glycerol
0.1 mg/mL BSA
The concentration of each component can be adjusted as needed to optimize the binding of the antibody to the DNA. For example, if the antibody is binding weakly, the concentration of the antibody or DNA can be increased. If the antibody is binding too strongly, the concentration of the salt or detergent can be increased.
It is also important to note that the binding buffer should be free of any components that could interfere with the binding of the antibody to the DNA. For example, detergents and chaotropic agents can disrupt the structure of the DNA, making it difficult for the antibody to bind. For optimizing the binding of an antibody to DNA in an EMSA, use a high-quality antibody.
The quality of the antibody can have a significant impact on the strength of the binding. Use a DNA probe that is the appropriate size for the antibody. The size of the DNA probe should be similar to the size of the epitope that the antibody recognizes. Use a high-quality DNA gel. The quality of the DNA gel can also affect the strength of the binding with a consistent protocol. This will help to ensure that the results are reproducible.
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