Blocking agents albumin

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. 26.4. Signals and backgrounds obtained when (a) albumin and (b) 1-hexanethiol are employed as blocking agents. Clocking agent = 2%, Cdna — liIM, Vdrop — 1 fd. Fimmob — d 0, I n n qh — 12 h. Reprinted from Ref. [9], Copyright 2005, with permission from Elsevier.

The choice of blocking buffer is sometimes critical for sensitive detection. Milk based blocking solutions are not recommended for use avidin-biotin system because milk contains biotin, which may directly cause competition with biotinylated antibody." Bovine Serum Albumin was not selected in this system in order to avoid the cross-reactivity. Therefore, gelatin was chosen as the blocking agent. 

Predominant protein blockers include bovine serum albumin (BSA), nonfat dry milk (NFDM), casein, and fish gelatin. NFDM, used at 0.1 0.5%, is inexpensive but preparations vary in quality. Some NFDM preparations contain histones that interfere with anti-DNA determinations or inhibitors of the biotin (strept)avidin interaction such as biotin itself. Casein is a chief component of NFDM and is often used alone as a blocking agent. 

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)]. 

There are two variables that affect the conjugation of TRITC to a protein, the amount of TRITC used, and the time allowed for the reaction to occur. Prior to labeling of affinity purified antibodies, the amount of TRITC and the time needed to achieve a labeling ratio between 1.5 and 1 TRITC labels per protein were determined. The TRITC labeled protein solutions used in all biosensor experiments were prepared at a protein concentration of 5 pg/mL in PBS containing 2.0 mg/mL Bovine Serum Albumin (BSA). The BSA was used as a blocking agent to block non-specific binding sites on the fiber. 

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