2.4. Membrane Blocking
In order to avoid background binding of a protein/antibody outside the physical boundary of SPOTed peptides, membranes are typically blocked overnight with an appropriate agent. The choice of blocking agent is also critical for the sensitivity of the antibody used for detection. We have tested different blocking agents, including 5% skimmed milk, 5% bovine serum albumin (BSA, Fisher Scientific, #70955) and 5% alkali-soluble casein (Novagen, distributed by Merck-Millipore, Felyham, UK, #70955). We obtained optimal signal to noise ratio using BSA. It is important to note that BSA is also preferred over milk when detecting phosphorylated proteins, although care must be taken to avoid BSA which contains tyrosine phosphorylations resulting in high background when used with anti-phosphotyrosine antibodies.