Why blocking is significant in Western Blot analysis? Membrane blocking in Western Blot is for the purpose of preventing the non-specific binding of antibodies including both primary antibody and secondary antibody to the membrane, so that the common problem of high background in western blot can be avoided.
Western blot blocking is a very important step of western blotting, as it prevents antibodies from binding to the membrane nonspecifically. Blocking is often made with BSA or nonfat dried milk diluted in TBST/PBST buffers. When it comes to these buffers, it is important to note that TBST is preferred with AP (Alkaline Phosphatase) labeled antibodies because PBS will interfere with the AP signal.
Nonfat dried milk is often preferred as it is inexpensive and widely available. However, milk proteins are not compatible with all detection labels, so care must be taken to choose the appropriate blocking solution. For example, BSA blocking solutions are preferred with biotin and AP antibody labels, and antiphosphoprotein antibodies, since milk contains casein, which is itself a phosphoprotein and biotin, thus interfering with the assay results.
3 - 5% non-fat milk or BSA (Bovine serum albumin) in 1X TBST or 1X PBST are commonly used as blocking buffers in Western Blot. But what are the pros and cons of non-fat milk and BSA?
Non-fat milk VS BSA as blocking agents
|Non-fat milk||• Cheap compared to BSA.
• Readily available and easy to prepare from powder.
|• Shouldn't be used to detect phosphorylated proteins.
• Should not be used if avidin-biotin detection systems are being used as milk contains biotin.
• Should be filtered to remove particulates binding to the membrane.
|BSA||• Good general blocking agent.
• Clearer results as it contains only one protein so less for the antibody to cross-react with.
• Works better with phospho-antibodies as albumin is a secreted protein and it tend to not be phosphorylated.
|• More expensive than milk.
• Should also be filtered to remove particulates.
• Not compatible with lectin probes as it contains carbohydrates that can increase background.
Incubate for 1 hour or more in membrane blocking solution at 4°C under agitation. Rinse the membrane for 5 seconds in TBST after the incubation of blocking.