SYPRO ORANGE AND SYPRO RED PROTEIN GEL STAINS - ONE-STEP FLUORESCENT STAINING OF DENATURING GELS FOR DETECTION OF NANOGRAM LEVELS OF PROTEIN

We have developed two new fluorescent dyes, SYPRO Orange protein gel s tain and SYPRO Red protein gel stain, to detect proteins in electropho retic gels. Stained protein bands can be excited by ultraviolet light at similar to 300 nm, or at visible wavelengths, with excitation maxim a of 472 nm for the Orange stain and 547 nm for the Red stain. Detecti on can be documented with sensitivity similar to that achieved with si lver stain, using standard UV transilluminators and Polaroid 667 black and white film, CCD cameras, or commercially available laser scanners . Staining with these dyes is noncovalent and is accomplished using a one-step procedure. Protein gels do not require fixation steps prior t o incubation with the dyes. Staining is complete 30 to 60 min followin g electrophoresis, with no destaining required. Staining can also be a ccomplished by including dye in the running buffer; in this case a bri ef one-step destaining procedure follows electrophoresis. The dyes app ear to bind to the detergent coat surrounding proteins in sodium dodec yl sulfate (SDS) denaturing gels; thus, staining in such gels is not s trongly selective for particular polypeptides. Fluorescent signals are relatively photostable, allowing multiple photographs of gels to be t aken without significant signal reduction. (C) 1996 Academic Press, In c.