F(ab') 2 fragments of antibodies are generated by pepsin digestion of whole IgG antibodies to remove most of the Fc region while leaving intact some of the hinge region. F(ab')2 fragments have two antigen-binding Fab portions linked together by disulfide bonds, and therefore are divalent (Figure 1). The average molecular weight is about 110 kDa. They are used for specific applications, such as to avoid binding of antibodies to live cells with Fc receptors or to Protein A or Protein G.
However, binding of primary antibodies to Fc receptors also may occur if they are whole IgG antibodies, creating background regardless of the form of the secondary antibody. To block whole IgG primary and secondary antibodies from binding to Fc receptors, incubate cells in buffer containing 5% normal serum from the host species of the secondary antibody. To prevent capping, endocytosis, and regeneration of Fc receptors on living cells, incubate at 4°C in buffer containing 5% normal serum with sodium azide added to inhibit metabolism.
Cautions: Never block with normal serum or IgG from the host species of the primary antibody when using a labeled secondary antibody. If immunoglobulins in the normal serum bind to the specimen of interest, they will be recognized by the labeled secondary antibody, resulting in higher background.
Bovine serum albumin (BSA) and dry milk, both commonly used for blocking, may contain bovine IgG. With the exception of Bovine anti-Goat IgG, many secondary antibodies such as anti-bovine, anti-goat, and anti-sheep will react strongly with bovine IgG. Therefore, use of BSA or dry milk for blocking or diluting these antibodies may significantly increase background and/or reduce antibody titer. For blocking, use normal serum (5% v/v) from the host species of the secondary antibody.
We also have a limited inventory of DyLight™ 488 / 549 / 594 / 647, Cy™2, Cy™5, and Texas Red® conjugated secondary antibodies.