AB fragments

Any heterocycle containing the OCH=CH moiety can in principle extrude the superfluous fragment and form oxirene, as illustrated for a five-membered ring in Scheme 105. Probably the most propitious AB fragment would be nitrogen, but the required 1,2,3-oxadiazole (123) is unknown (see Chapter 4.21), probably because of ready valence tautomerization to diazoethanal (Scheme 106) (this approach has been spectacularly successful with the sulfur analogue of (2) (8UA486)). The use of (123) as an oxirene precursor is thus closely linked to the important diazo ketone decompositions discussed in Section 5.05.6.3.4(f). 

In most cases, however, blocking endogenous Fc receptors is nothing more than a ritual. There are no persuasive studies demonstrating that the blocking serum step is really necessary. In any case, the use of labeled F(ab )2 or F(ab) fragments for the secondary antibodies in place of the whole IgG form of the secondary antibody eliminates the need to block endogenous Fc receptors (see Sect. 4.3.2). 

Peelers, K., De Wilde, C., and Depicker, A. (2001). Highly efficient targeting and accumulation of a F(ab) fragment within the secretory pathway and apoplast of Arabidopsis thaliana. Ear. J. Biochem. 268 4251 260. 

Key Words Antibody (Ab) fragment antigen binding (Fab) fragment variable (Fv) single-chain Fv (scFv) variable part of heavy chain variable part of light chain wild type. 

Figure 2. Inhibition by lectins of the binding of, 2>I F(ab) fragments of anti-H-2.4 IgG to lymphocytes. (A) M. pomifera agglutinin (Q) concanavalin A (Q) W. floribunda mitogen (O) S. japonica agglutinin.

Figure 3. Scatchard plot of the binding of t2SI F(ab) fragments of anti-H-2.4 IgG to lymphocytes...

Binding of the Fc portion of Ig by Fc receptors on the cell membrane of macrophages, monocytes, granulocytes and some lymphocytes. Use F(ab )2 or F(ab) fragments for the primary and secondary antibodies rather than intact antibodies. Add detergent to the wash buffer. 115-121... 

It is necessary to test concentrations for each new antibody, whether primary or secondary. For blocking before application of the primary, use serum from the same species that your secondary is made in e.g., if your secondary is made in a rabbit, block with normal rabbit serum but if your secondary is made in a pig use swine serum. For secondary antibodies it is advisable to use F(ab) fragments (to minimize cross reaction with FcR on macrophages or B-cells). 

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