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 stain and SYPRO Red protein gel stain, to detect proteins in electrophoretic gels. Stained protein bands can be excited by ultraviolet light at approximately 300 nm, or at visible wavelengths, with excitation maxima of 472 nm for the Orange stain and 547 nm for the Red stain. Detection can be documented with sensitivity similar to that achieved with silver 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 to incubation with the dyes. Staining is complete 30 to 60 min following electrophoresis, with no destaining required. Staining can also be accomplished by including dye in the running buffer; in this case a brief one-step destaining procedure follows electrophoresis. The dyes appear to bind to the detergent coat surrounding proteins in sodium dodecyl sulfate (SDS) denaturing gels; thus, staining in such gels is not strongly selective for particular polypeptides. Fluorescent signals are relatively photostable, allowing multiple photographs of gels to be taken without significant signal reduction.