Blocking agents nonspecific binding

Add a blocking agent, such as a non-relevant protein (e.g., BSA) to a final concentration of 1 percent to mask any nonspecific binding sites and to couple with any remaining reactive groups on the silica particle surface. This is important especially if a limiting amount of antibody was initially reacted with the particles in step 5. React for 30 minutes to 1 hour at room temperature. 

The proper choice of an application buffer can help to minimize any nonspecific binding due to undesired sample components. For example, coulombic interactions between solutes and the support can often be decreased by altering the ionic strength and pH of the application buffer. In addition, surfactants and blocking agents (e.g., Triton X-100, Tween-20, bovine serum albumin, and gelatin) may be added to the buffer to prevent nonspecific retention of solutes on the support or affinity ligand. 

Fig. 3.7 Spectral changes caused by thrombin (10 nM) binding to NSTBA sensor. The draninant peaks can be assigned to DNA vibrational modes with a lesser contribution from protein vibrational modes. Controls with BSA (10 mg/ml), msulin 600 nM and 1% human serum show no nonspecific protein binding. Lower three traces show spectra of NS, NS plus blocking agent (mercaptohexanol) and the fully assembled NSTBA sensor. Spectra are averages of 10 acquisitions...

Using proteins as an example, the purpose of blocking is to cover sites on the membrane that do not contain protein so you get nonspecific binding. Proteins adhere to nitrocellulose. If you block the other areas to prevent staining other compounds then you do not need to remove these other compounds. Some good blocking agents for proteins are nonfat dry milk, bovine serum albumin (BSA), and polyvinylpyrrolidone (PVP). 

Blocking agent is used to suppress nonspecific binding of antibodies. A concentrated solution (200 mg/ml) of bovine serum albumin (BSA) in PBS is used. The solution is diluted 1 1 with PBS and centrifuged for 5 min at 10,000 g before use to remove any debris [Organon, Oss, The Netherlands cat. 81537 (Boseral 20T)]. 

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