An improved, luminescent europium-based stain for detection of electroblotted proteins on nitrocellulose or polyvinylidene difluoride membranes

SYPRO Rose Plus protein blot stain is an improved europium-based metal chel ate stain for the detection of proteins on nitrocellulose and poly(vinylide ne difluoride) (PVDF) membranes. Staining is achieved without covalently mo difying the proteins. The stain may be excited with a 254 nm (UV-C), 302 nm (UV-B), or 365 nm (UV-A) light source and displays a sharp emission maximu m at 612 nm. The emission peak has a full width at half-maximum of only 8 n m. The stain exhibits exceptional photostability, allowing long exposure ti mes for maximum sensitivity. Since the dye is composed of a europium comple x, it has a long emission lifetime, potentially allowing time-resolved dete ction, greatly reducing background fluorescence. Proteins immobilized to a nitrocellulose or PVDF membrane by electroblotting, dot-blotting, or Vacuum slot-blotting are incubated with SYPRO Rose Plus protein blot stain for 15 -30 min. Membranes are rinsed briefly, visualized with UV epi-illumination and the luminescence of the europium dye is measured using a 490 nm long-pa ss or 625 +/- 15 nm band-pass filter in combination with a conventional pho tographic or charge-coupled device (CCD) camera system. Alternatively, the dye may be visualized using a xenon-are illumination source. The stain is r eadily removed from proteins by incubating membranes at mildly alkaline pH. The reversibility of the protein staining procedure allows for subsequent biochemical analyses, such as immunoblotting and biotin-streptavidin detect ion using colorimetric, direct fluorescence or fluorogenic visualization me thods.