In order to determine the distance between the antigen-binding site and the fragment crystallizable (Fc) region of an antibody using single-molecule Förster Resonance Energy Transfer, dyes must be attached to these locations. To accomplish this, the variable regions of the light and heavy chains of an antibody can be modified and a dye-reactive amino acid introduced into the Fc region. Initial attempts to alter the variable light chain (VL) gene included trying to ligate a 350bp variable gene into an 11,000bp plasmid. When these attempts were unsuccessful, the variable light chain gene was ligated into a commercially available 3500bp plasmid.

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