Labeling antibodies with pHrodo green and red amine-reactive reagents

The pHrodo Red amine-reactive ester was conjugated to the EE1 antibody as a marker of early endosomes. The pHrodo Green amine-reactive ester was conjugated to RAB7 as a marker of late endosomes. The pHrodo Green amine-reactive ester was conjugated with a monoclonal anti-GRP94 antibody. GRP94 is a resident protein of the endoplasmic reticulum that is induced by accumulation of unfolded proteins and associates transiently with various newly synthesized secretory and membrane proteins. The pHrodo Red amine-reactive ester was conjugated with a monoclonal anti-CDF4 antibody (golgin 97 mon 3h811o) and an anti-mucin antibody (5ac) as Golgi markers. The antibodies were purified before labeling by dialysis with PBS (pH 7.2–7.5) to remove low molecular weight components (i.e., desalting). Labeling was then performed according to the manufacturer instructions then purified by extensive dialysis. The relative efficiency of the labeling reactions for each antibody was determined by measuring the absorbance of the dyes at their excitation maximum and protein absorbance at 280 nm.