PBXL-1: A New Fluorochrome Applied to Detection of Proteins on Membranes

Abstract

An easy, sensitive and direct fluorescent immunodetection method for proteins is described using the new fluorochrome PBXL™-1 imaged with the FMBIO® II Laser Scanning Imaging System. PBXL-1 is derived from a protein supra-molecular complex that contains a large number of chromophores. This complex, the phycobilisome, is extracted from a red alga then chemically stabilized to allow its use in specific binding assays. PBXL-1 was crosslinked to goat anti-rabbit IgG or streptavidin with heterobifunctional cross-linkers. The detection limit of PBXL-1 was determined by applying it on nitrocellulose membranes then imaging the membrane using an ytterbium aluminum garnet (YAG) laser. Evaluation of PBXL-1 sensitivity in a specific binding assay was tested on streptavidin/biotin and an antibody system. PBXL-1 provides high sensitivity in direct fluorescent applications due to a physical amplification of signal (i.e., a large number of fluorophores per binding event). PBXL-1 provides a linear response over two orders of magnitude while providing sub-amol sensitivity, indicating broad applicability for detection of a variety of targets. To our knowledge, this is the most sensitive direct fluorescent detection method available.