You can either use BSA or non-fat milk to block non-specific sites within the membrane where your proteins are in order to avoid the binding of the primary antibody to non desired sites. It also reduces the background of the secondary following the same principle. The amount of BSA or milk used is user and antibody-dependent.
Francisco Javier Rodríguez Baena.. Is the concentration of BSA (1%) specific for primary antibodies or is it derived after standardising the experiments?
1% BSA is enough to block unspecific interactions and reduce the background for most antibodies independent of the origin. But if your background is high, you could try to increase the concentration of BSA
Julio Raúl Fernández Massó.. Thank you for sharing sir. My frind has an opinion that 15 is used so that the antibody dosent go under stress, because their quantity is very less in a marquee solution. Do you agree or have some other opinion?
Hi again Ashwin, the 1% it's not specific for the primary antibodies. In a general protocol for WB you will find that people use to block with a 3% BSA or 3-5% non-fat powdered milk and then reduce that amount to 1% because you're not blocking anymore but the presence of these proteins within the antibody solution helps to preserve the own antibody as well as the binding would be even more specific.
I want to add one more consideration. If you are going to use the blot for phosphoserin, phosphothreonine or phosphotyrosine antibodies you must avoid using powdered milk because milk has phosphorylated casein and membrane will be useless for that western blot detection. You must use in this case, 3% BSA. This is a common mistake.