Protein detection

   SYPRO Ruby protein blot stain is a new, luminescent metal chelate stain composed of ruthenium as part of an organic complex that interacts noncovalently with proteins. Fluorescence emission of the dye is approximately 618 nm. SYPRO Ruby protein blot stain allows sensitive fluorescence detection of proteins on a variety of solid-phase supports, including polyvinylidene difluoride membranes, nitrocellulose membranes, and glass. The staining procedure requires only 15 min and no detergent blocking step or fixation is required to selectively visualize proteins. After staining, blots are simply rinsed in water and visualized using a standard UV light box, or any of a variety of laser-based gel scanners [12].

    Fluorescence excitation/emission spectra of SYPRO Ruby protein blot stain demonstrate that the dye is optimally excited with 302 or 470 nm light (Fig. 1). SYPRO Ruby dye has a relatively broad emission maximum centered at 618 nm [12].

Fig. 1. Fluorescence excitation/emission spectra of SYPRO Ruby protein blot stain. (A) Excitation maxima at 300 and 470 nm with emission set at 618 nm. (B) Emission maximum at 618 nm with excitation set at 470 nm.

   Typically, 302 nm epi- or transillumination as well as excitation with 450 to 490 nm visible light sources readily allows detection of SYPRO Ruby protein blot stain. Surprisingly, we discovered that solid-state yttrium–aluminum–garnet (YAG) laser sources with emission maxima centered at 532 nm are also suitable for sensitive visualization of SYPRO Ruby dye, even though this excitation wavelength is far from the dye’s optimum. 490 to 610 nm long-pass or 490 to 650 nm band-pass emission filters are suitable for visualizing the dye [12].

   Several methods for fluorescent detection of proteins on nitrocellulose or polyvinylidene difluoride (PVDF) membranes have been devised utilizing fluorescamine, fluorescein isothiocyanate, dichlorotriazinylaminofluorescein, or dansyl chloride; the cited methods include the need to covalently modify proteins. One recently developed high sensitivity detection method uses a nonenzymatic, chemiluminescent reaction consisting of bis(2,4,6- trichlorophenyl)oxylate and hydrogen peroxide for visualization of electroblotted or dot-blotted proteins covalently labeled with 2-methoxy-2,4-diphenyl-3(2H)- furanone (MDPF) . The direct fluorescence method allows detection of 5–10 ng of protein and is compatible with chemiluminescent immunodetection. However, proteins labeled with MPDF must be viewed using wet PVDF membranes because fluorescence signal decreases 500-fold upon drying. If proteins are to be used for N-terminal sequencing, staining must be performed with a suboptimal dye concentration for a short period of time so that all primary amines are not consumed in the reaction. Under these conditions the fluorescent stain is no more sensitive than other staining procedures [12].

    A readily reversible, luminescent metal chelate stain consisting of europium complexed to bathophenanthroline disulfonate has been shown to be useful for detection of low-nanogram quantities of proteins immobilized on nitrocellulose or PVDF membranes. The stain, now commercialized as SYPRO Rose protein blot stain (Molecular Probes), is highly resistant to photobleaching and compatible with popular downstream biochemical characterization procedures. The stain can be excited at 302 nm using standard UV epi-illumination and displays emission maxima at 590 and 615 nm [12].

   Two benefits are higher detection sensitivity, and minimal cross-reactivity with nucleic acids. Since the stain does not detect nucleic acids, it is appropriate for quantitation of nucleic acid binding proteins. Limitations of SYPRO Ruby protein blot stain relative to bathophenanthroline disulfonate/europium stain include a fairly broad emission peak, which could complicate multicolor visualization procedures and greater difficulty in removing the stain from bound protein, an important attribute in some quality control environments such as detection of bound protein on the surface of contact lenses. SYPRO Ruby protein blot stain offers additional advantages such as a wide linear dynamic range and full compatibility with modern microchemical techniques. SYPRO Ruby protein blot stain may be useful in the quality control of high-density protein microarrays used for high-throughput screens of gene expression and receptor ligand interactions [12].