PBXL-1: a new fluorochrome applied to detection of proteins on membranes.
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 cross-linked 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.
Morseman
J P
JP
Martek Biosciences, Columbia, MD, USA.
Moss
M W
MW
Zoha
S J
SJ
Allnutt
F C
FC
D000818:Animals; D019921:Biotinylation; D015153:Blotting, Western; D002417:Cattle; D019085:Fluorescent Antibody Technique, Direct; D005456:Fluorescent Dyes; D006041:Goats; D007074:Immunoglobulin G; D045524:Phycobilisomes; D011506:Proteins / Q000032:analysis*; D011817:Rabbits; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000032:analysis; D019809:Streptavidin
D005456:Fluorescent Dyes; D007074:Immunoglobulin G; D045524:Phycobilisomes; D011506:Proteins; D012710:Serum Albumin, Bovine; D019809:Streptavidin