Anyone who does the Western blot routinely knows the feeling: You test new antibodies and are filled with anticipation when your film comes out of X-Omat. A few seconds later, your expectations are destroyed because you realize your sample is lost because of the bad blot. Non-specific bands and high backgrounds in the West can be the main source of frustration on the bench. To get more details you can search about some companies like bosterbio that provide western blot services.
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The tips below are designed to help clean back the background on your notes.
1. Optimize your antibody concentration
Adding too many antibodies can cause numbers with high backgrounds and non-specific bands. Check the suggested dilution for your main antibodies and consider reducing the concentration of primary antibodies. Using excess secondary antibodies may also produce obstacles with high backgrounds. Blot control without primary antibodies can help determine whether your secondary antibodies contribute to non-specific signals.
2. Be sure to block
Blocking is very important for clean stains. Blocking agents such as non-fat dry milk or BSA occupy non-specific binding sites that produce lower backgrounds. Check to see if there are the preferred blocking agents and concentrations for your antibodies. 1-5% blocking solutions are usually recommended.
Block for one hour at room temperature or overnight at 4 degrees with agitation. Blocking solutions must be made fresh because bacterial growth can cause high background. When making a choice between blocking with milk and BSA, remember that milk contains casein, phosphoprotein which can increase the background when used with phospho-specific antibodies.