A Simple and Rapid Purification Procedure for the Preparation of Tetramethylrhodamine-Labeled Antibodies

Abstract

A conjugate preparation of antibody (Fab) and tetramethylrhodamine (TMR) generally adsorbs free TMR which is very difficult to remove because of its strong hydrophobic binding. On the basis of criteria such as sodium dodecyl sulfate (SDS) gel electrophoresis and staining of the plasma membrane of live cells, we found that simple extraction with n-butyl alcohol or iso-amyl alcohol could remove the contaminating free dye. The procedure is especially useful when one needs to prepare conjugates with low nonspecific binding for the study of lateral diffusion of cell membrane-associated antigens.