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A new formulation of the small-molecule organic fluorophore, Pro-Q® Diamond dye, has been developed that permits rapid and simple detection of phosphoproteins directly on polyvinylidene difluoride (PVDF) or nitrocellulose membranes (electroblots). Protein samples are first separated by electrophoresis and then electroblotted to membranes, stained and destained, in an analogous manner as typically performed with Amido Black or Ponceau S dye staining of total protein profiles. After staining, blots are imaged using any of a variety of laser-based gel scanners, xenon-arc lamp-based gel scanners or charge-coupled device (CCD) camera-based imaging devices equipped with UV trans- or epi-illumination. The uncomplicated and reliable staining protocol delivers results in as little as 1 h and the limit of detection for the stain is typically 2-4 ng of phosphoprotein with a linear dynamic range of approximately 15-fold. Compared with traditional radiolabeling and antibody-based approaches, the new method offers significant advantages, including avoidance of radioactivity, no need for expensive antibodies, no requirement for blocking unoccupied sites on the membrane with protein or detergent solutions, no sequence context-specific binding to phosphorylated amino acid residues and the ability to analyze the native, steady-state phosphorylation of proteins obtained directly from tissue specimens or body fluids. Pro-Q Diamond dye binds directly and exclusively to the phosphate moiety, allowing it to detect the broadest spectrum of phosphorylated proteins possible. The stain binds noncovalently to phosphoproteins and is thus fully compatible with matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) or Edman sequencing. The blot stain is also compatible with standard colorimetric, fluorogenic, and chemiluminescent detection techniques employed in immunoblotting.

Pro-Q Emerald 488 glycoprotein stain reacts with periodic acid-oxidized carbohydrate groups, generating a bright green-fluorescent signal on glycoproteins. The stain permits detection of less than 5-18 ng of glycoprotein per band, depending upon the nature and the degree of protein glycosylation, making it roughly 8-16-fold more sensitive than the standard colorimetric periodic acid-Schiff base method using acidic fuchsin dye (pararosaniline). The green-fluorescent signal from Pro-Q Emerald 488 stain may optimally be visualized using charge-coupled device/xenon arc lamp-based imaging systems or 470-488 nm laser-based gel scanners. Though glycoprotein detection may be performed on transfer membranes, direct detection in gels avoids electroblotting and the specificity of staining is better in gels. After detecting glycoproteins with Pro-Q Emerald 488 dye, total protein profiles may subsequently be detected using SYPRO Ruby protein gel stain. Using computer-assisted registration techniques, images may then be merged to generate differential display maps.

A dichromatic method for measuring the specific activity of beta-glucuronidase from complex cell homogenates or partially purified protein fractions is presented. Dual fluorescence is achieved by using the green emitting fluorogenic substrate ELF 97 beta-D-glucuronide to detect beta-glucuronidase activity, followed by the red emitting SYPRO Ruby protein gel stain or SYPRO Ruby IEF gel stain to detect the remaining proteins in the electrophoretic profile. Both ELF 97 alcohol, the highly fluorescent hydrolytic product generated from the enzyme substrate, and the SYPRO Ruby total protein stains are maximally excited by ultraviolet illumination. ELF 97 alcohol emits maximally at 525 nm while the SYPRO Ruby dyes emit maximally at 610 nm. Since ELF 97 beta-glucuronide is a precipitating substrate, it allows precise localization of beta-glucuronidase activity with minimal band diffusion. The staining method is simple and direct, without the requirement for ancillary coupling reactions. Dichromatic protein detection is demonstrated after sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis, carrier ampholyte-mediated isoelectric focusing or two-dimensional gel electrophoresis.

SYPRO Ruby dye is a permanent stain comprised of ruthenium as part of an organic complex that interacts noncovalently with proteins. SYPRO Ruby Protein Gel Stain provides a sensitive, gentle, fluorescence-based method for detecting proteins in one-dimensional and two-dimensional sodium dodecyl sulfate-polyacrylamide gels. Proteins are fixed, stained from 3h to overnight and then rinsed in deionized water or dilute methanol/acetic acid solution for 30 min. The stain can be visualized using a wide range of excitation sources commonly used in image analysis systems including a 302 nm UV-B transilluminator, 473 nm second harmonic generation (SHG) laser, 488 nm argon-ion laser, 532 nm yttrium-aluminum-garnet (YAG) laser, xenon arc lamp, blue fluorescent light bulb or blue light-emitting diode (LED). The sensitivity of SYPRO Ruby Protein Gel Stain is superior to colloidal Coomassie Brilliant Blue (CBB) stain or monobromobimane labeling and comparable with the highest sensitivity silver or zinc-imidazole staining procedures available. The linear dynamic range of SYPRO Ruby Protein Gel stain extends over three orders of magnitude, which is vastly superior to silver, zinc-imidazole, monobromobimane and CBB stain. The fluorescent stain does not contain superfluous chemicals (formaldehyde, glutaraldehyde, Tween-20) that frequently interfere with peptide identification in mass spectrometry. While peptide mass profiles are severely altered in protein samples prelabeled with monobromobimane, successful identification of proteins by peptide mass profiling using matrix-assisted laser desorption/ionization mass spectrometry was easily performed after protein detection with SYPRO Ruby Protein Gel stain.

SYPRO Tangerine stain is an environmentally benign alternative to conventional protein stains that does not require solvents such as methanol or acetic acid for effective protein visualization. Instead, proteins can be stained in a wide range of buffers, including phosphate-buffered saline or simply 150 mM NaCl using an easy, one-step procedure that does not require destaining. Stained proteins can be excited by ultraviolet light of about 300 nm or with visible light of about 490 nm. The fluorescence emission maximum of the dye is approximately 640 nm. Noncovalent binding of SYPRO Tangerine dye is mediated by sodium dodecyl sulfate (SDS) and to a lesser extent by hydrophobic amino acid residues in proteins. This is in stark contrast to acidic silver nitrate staining, which interacts predominantly with lysine residues or Coomassie Blue R, which in turn interacts primarily with arginine and lysine residues. The sensitivity of SYPRO Tangerine stain is similar to that of the SYPRO Red and SYPRO Orange stains - about 4-10 ng per protein band. This detection sensitivity is comparable to colloidal Coomassie blue staining and rapid silver staining procedures. Since proteins stained with SYPRO Tangerine dye are not fixed, they can easily be eluted from gels or utilized in zymographic assays, provided that SDS does not inactivate the protein of interest. This is demonstrated with in-gel detection of rabbit liver esterase activity using alpha-naphthyl acetate and Fast Blue BB dye as well as Escherichia coli beta-glucuronidase activity using ELF-97 beta-D-glucuronide. The dye is also suitable for staining proteins in gels prior to their transfer to membranes by electroblotting. Gentle staining conditions are expected to improve protein recovery after electroelution and to reduce the potential for artifactual protein modifications such as the alkylation of lysine and esterification of glutamate residues, which complicate interpretation of peptide fragment profiles generated by mass spectrometry.

SYPRO Ruby IEF Protein Gel Stain is an ultrasensitive, luminescent stain optimized for the analysis of protein in isoelectric focusing gels. Proteins are stained in a ruthenium-containing metal complex overnight and then rinsed in distilled water for 2 h. Stained proteins can be excited by ultraviolet light of about 302 nm (UV-B transilluminator) or with visible light of about 470 nm. Fluorescence emission of the dye is maximal at approximately 610 nm. The sensitivity of the SYPRO Ruby IEF protein gel stain is superior to colloidal Coomassie blue stain and the highest sensitivity silver staining procedures available. The SYPRO Ruby IEF protein gel stain is suitable for staining proteins in nondenaturing or denaturing carrier ampholyte isoelectric focusing and immobilized pH gradient gel electrophoresis. The stain is compatible with N,N'-methylenebisacrylamide or piperazine diacylamide cross-linked polyacrylamide gels as well as with agarose gels and high tensile strength Duracryl gels. The stain does not contain extraneous chemicals (formaldehyde, glutaraldehyde, Tween-20) that frequently interfere with peptide identification in mass spectrometry. Successful identification of stained proteins by peptide mass profiling is demonstrated.