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Information on blocking for western blots.
Western Blot Blocking
Membrane Blocking in Western Blotting Introduction Unfortunately, since membranes are chosen for their ability to bind to proteins, and antibodies are proteins, there is usually a lot of non-specific binding which affects the quality of western blots. Specific primary and secondary antibodies may bind non-specifically to the membrane as they are proteins. The problem is detected during detection, where non-specific bands or general detection makes detection of specific bands impossible. How to Block Non-specific Interactions in Western Blots? In western blots, blocking using proteins is conducted to "block" all spots on the membrane where antibodies may bind non-specifically to. Blocking Methods in Western Blotting In western blotting, common agents such as Bovine Serum Albumin (BSA) or 1-5% non-fat dry milk (NFDM) are used before incubating membranes in primary and secondary antibodies. Both of these are inexpensive blocking agents. Small amounts of of detergent such as Tween 20 can also be added. Variations to Western Blot Blocking Blocking may be also done during incubations of primary and secondary antibodies to improve the quality of the western blot. Conclusion for Blocking In conclusion, the protein in the blocking agents (albumin or other proteins in milk) attach non-specifically to the membrane and block antibody non-specific binding. Therefore, when the membrane is incubated with antibody, there is no attachment sites for binding. The end result of blocking is a reduction in noise coming from non-specific binding and hence allowing cleaner results and the elimination of false positives.
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