title: An improved, luminescent europium-based stain for detection of electroblotted proteins on nitrocellulose or polyvinylidene difluoride membranes.
abstract: SYPRO Rose Plus protein blot stain is an improved europium-based metal chelate stain for the detection of proteins on nitrocellulose and poly(vinylidene difluoride) (PVDF) membranes. Staining is achieved without covalently modifying 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 maximum at 612 nm. The emission peak has a full width at half-maximum of only 8 nm. The stain exhibits exceptional photostability, allowing long exposure times for maximum sensitivity. Since the dye is composed of a europium complex, it has a long emission lifetime, potentially allowing time-resolved detection, 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-pass or 625 +/- 15 nm band-pass filter in combination with a conventional photographic or charge-coupled device (CCD) camera system. Alternatively, the dye may be visualized using a xenon-arc illumination source. The stain is readily 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 detection using colorimetric, direct fluorescence or fluorogenic visualization methods.
authors.affiliation: Molecular Probes, Inc, Eugene, OR 97402, USA.
authors.forename: C
authors.initials: C
authors.lastname: Kemper
authors.forename: K
authors.initials: K
authors.lastname: Berggren
authors.forename: Z
authors.initials: Z
authors.lastname: Diwu
authors.forename: W F
authors.initials: WF
authors.lastname: Patton
mesh_terms: D015153:Blotting, Western; D003101:Collodion; D004396:Coloring Agents; D004586:Electrophoresis / Q000379:methods; D005063:Europium; D015151:Immunoblotting; D008163:Luminescent Measurements; D011145:Polyvinyls; D011506:Proteins / Q000032:analysis*; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D013194:Staining and Labeling / Q000379:methods; D005465:Fluorocarbon Polymers
chemical_list: D003101:Collodion; D004396:Coloring Agents; D005063:Europium; D011145:Polyvinyls; D011506:Proteins; C436863:SYPRO Rose Plus; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers