23] Application of green fluorescent protein-protein a fusion protein to western blotting

Abstract

Publisher Summary Green fluorescent protein (GFP) that is isolated from the jellyfish Aequorea victoria noncatalytically produces an intense and stable greenish fluorescence. Aequorea GFP maximally absorbs blue light at 395 nm and emits green light with a peak at 509 nm. GFP is a protein of 238 amino acids with a molecular mass of 27-30 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The hexapeptide segment, beginning at residue 64, functions as a fluorescent chromophore formed on cyclization of the residues Ser· dehydro-Tyr-Gly within the hexapeptide, by posttranslational modification. GFP has a unique structure and interesting physical properties—for example, high stability to denaturing reagents or proteases. GFP fluorescence occurs without cofactors and this property allows GFP fluorescence in nonnative organisms in which GFP is expressed. Although GFP is relatively large to serve as a fusion tag, GFP-tagged proteins retain their original functions in many cases. Therefore, GFP has been used as a reporter for gene expression, as a tracer of cell lineage, and as a fusion tag to investigate protein localization and secretion systems in vivo. The reports of GFP fusions to protein A and to streptavidin, and of a simple immunoassay system using a GFP tag also indicate a wide range of in vitro applications. This chapter discusses the construction of a protein A-GFP fusion (PA-GFP) and its use as a labeled antibody-specific ligand in immunoblotting. Immunoblotting requires a labeled antibody or antibody-specific ligand (such as protein A) and a system specifically for detection of the label. Labeling reagents frequently used have been enzymes, such as peroxidase, alkaline phosphatase, and β-galactosidase; gold particles, radioisotopes, and fluorochromes have also been used.