Optimization of Fluorescence Probe Preparation for R-Phycoerythrin in Porphyra yezoensis

Abstract:

We optimized the chemical conjugation between R-phycoerythrin and antibody. First, the R-PE (R-phycoerythrin) was derived with beterobifunctional reagent SPDP(N-succinimidyl-3-2-pyridyldithio propionate) and antibody was thiolated with DTT(dithiothreitol). Second, we determined the effects of the different molar ratio of SPDP to R-PE for the derivation and DTT to IgG for the thiolation. The results showed that the optimum molar ratio of SPDP to R-PE for derivation was 40:1, and DTT to IgG for thiolation was 500:1. The conjugation between R-phycoerythin and antibody was further done for fluorescence probe preparation. R-phycoerythin was conjugated with IgG and formed a probe complex by whole wavelength scanning, electrophoresis (Native-PAGE) and fluorescence microscope observation.