Kiera Berggren

Background-free, high sensitivity staining of proteins in one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gels using a luminescent ruthenium complex

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.

A comparison of silver stain and SYPRO Ruby Protein Gel Stain with respect to protein detection in two-dimensional gels and identification by peptide mass profiling

Proteomic projects are often focused on the discovery of differentially expressed proteins between control and experimental samples. Most laboratories choose the approach of running two‐dimensional (2‐D) gels, analyzing them and identifying the differentially expressed proteins by in‐gel digestion and mass spectrometry. To date, the available stains for visualizing proteins on 2‐D gels have been less than ideal for these projects because of poor detection sensitivity (Coomassie blue stain) or poor peptide recovery from in‐gel digests and mass spectrometry (silver stain), unless extra destaining and washing steps are included in the protocol. In addition, the limited dynamic range of these stains has made it difficult to rigorously and reliably determine subtle differences in protein quantities. SYPRO Ruby Protein Gel Stain is a novel, ruthenium‐based fluorescent dye for the detection of proteins in sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) gels that has properties making it well suited to high‐throughput proteomics projects. The advantages of SYPRO Ruby Protein Gel Stain relative to silver stain demonstrated in this study include a broad linear dynamic range and enhanced recovery of peptides from in‐gel digests for matrix assisted laser desorption/ionization‐time of flight (MALDI‐TOF) mass spectrometry.

Rapid and simple single nanogram detection of glycoproteins in polyacrylamide gels and on electroblots.

The fluorescent hydrazide, Pro‐Q Emerald 300 dye, may be conjugated to glycoproteins by a periodic acid Schiff’s (PAS) mechanism. The glycols present in glycoproteins are initially oxidized to aldehydes using periodic acid. The dye then reacts with the aldehydes to generate a highly fluorescent conjugate. Reduction with sodium metabisulfite or sodium borohydride is not required to stabilize the conjugate. Though glycoprotein detection may be performed on transfer membranes, direct detection in gels avoids electroblotting and glycoproteins may be visualized within 2–4 h of electrophoresis. This is substantially more rapid than PAS labeling with digoxigenin hydrazide followed by detection with an antidigoxigenin antibody conjugate of alkaline phosphatase, or PAS labeling with biotin hydrazide followed by detection with horseradish peroxidase or alkaline phosphatase conjugates of streptavidin, which require more than eight hours to complete. Pro‐Q Emerald 300 dye‐labeled gels and blots may be post‐stained with SYPRO Ruby dye, allowing sequential two‐color detection of glycosylated and nonglycosylated proteins. Both fluorophores are excited with mid‐range UV illumination. Pro‐Q Emerald 300 dye maximally emits at 530 nm (green) while SYPRO Ruby dye maximally emits at 610 nm (red). As little as 300 pg of α1‐acid glycoprotein (40% carbohydrate) and 1 ng of glucose oxidase (12% carbohydrate) or avidin (7% carbohydrate) are detectable in gels after staining with Pro‐Q Emerald 300 dye. Besides glycoproteins, as little as 2–4 ng of lipopolysaccharide is detectable in gels using Pro‐Q Emerald 300 dye while 250–1000 ng is required for detection with conventional silver staining. Detection of glycoproteins may be achieved in sodium dodecyl sulfate‐polyacrylamide gels, two‐dimensional gels and on polyvinylidene difluoride membranes.

An improved formulation of SYPRO Ruby protein gel stain: Comparison with the original formulation and with a ruthenium II tris (bathophenanthroline disulfonate) formulation

SYPRO Ruby protein gel stain is compatible with a variety of imaging platforms since it absorbs maximally in the ultraviolet (280 nm) and visible (470 nm) regions of the spectrum. Dye localization is achieved by noncovalent, electrostatic and hydrophobic binding to proteins, with signal being detected at 610 nm. Since proteins are not covalently modified by the dye, compatibility with downstream proteomics techniques such as matrix‐assisted laser desorption/ionisation‐time of flight mass spectrometry is assured. The principal limitation of the original formulation of SYPRO Ruby protein gel stain, is that it was only compatible with a limited number of gel fixation procedures. Too aggressive a fixation protocol led to diminished signal intensity and poor detection sensitivity. This is particularly apparent when post‐staining gels subjected to labeling with other fluorophores such as Schiffs base staining of glycoproteins with fluorescent hydrazides. Consequently, we have developed an improved formulation of SYPRO Ruby protein gel stain that is fully compatible with commonly implemented protein fixation procedures and is suitable for post‐staining gels after detection of glycoproteins using the green fluorescent Pro‐Q Emerald 300 glycoprotein stain or detection of β‐glucuronidase using the green fluorescent ELF 97 β‐D‐glucuronide. The new stain formulation is brighter, making it easier to manually excise spots for peptide mass profiling. An additional benefit of the improved formulation is that it permits staining of proteins in isoelectric focusing gels, without the requirement for caustic acids.

Fluorescence detection of proteins in sodium dodecyl sulfate-polyacrylamide gels using environmentally benign, nonfixative, saline solution

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 α‐naphthyl acetate and Fast Blue BB dye as well as Escherichia coli β‐glucuronidase activity using ELF‐97 β‐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.

Comparison of three different fluorescent visualization strategies for detecting Escherichia coli ATP synthase subunits after sodium dodecyl sulfate-polyacrylamide gel electrophoresis

The correlation between protein molecular weight and the number of lysine or basic amino acid residues was found to be high for broad range molecular weight standards, subunits of Escherichia coli F1F0‐ATP synthase and the translated open reading frame of E. coli. A relatively poor correlation between protein molecular weight and the number of cysteine residues was observed in all cases. The ability of amine‐reactive, thiol‐reactive and basic amino acid‐binding fluorophores to detect the eight subunits of F1F0‐ATP synthase complex was assessed using 2‐methoxy‐2,4‐diphenyl‐3(2H)‐furanone (MDPF), monobromobimane (MBB) and SYPRO Ruby protein gel stain, respectively. Though experimentally none of the fluorophores provided accurate estimates of the subunit stoichiometry of this complex, MDPF and SYPRO Ruby protein gel stain were capable of semiquantitative detection of every subunit. MBB, however, failed to detect subunits a, b and c of the hydrophobic F0 complex, as well as subunit ε of the F1 complex. All three fluorescent detection procedures permitted subsequent identification of representative subunits by peptide mass profiling using matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS). The use of thiol‐reactive fluorophores for the global analysis of protein expression profiles does not appear to be advisable as a significant number of proteins have few or no cysteine residues, thus escaping detection.

An improved, luminescent europium‐based stain for detection of electroblotted proteins on nitrocellulose or polyvinylidene difluoride membranes

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.

Green/red dual fluorescence detection of total protein and alkaline phosphate-conjugated probes on blotting membranes.

A two‐color fluorescence detection method is described based upon covalently coupling the succinimidyl ester of BODIPY FL‐X to proteins immobilized on poly(vinylidene difluoride) (PVDF) membranes, followed by detection of target proteins using the fluorogenic substrate 9H‐(1,3‐dichloro‐9,9‐dimethylacridin‐2‐one‐7‐yl(DDAO)‐phosphate in combination with alkaline‐phosphatase‐conjugated reporter molecules. This results in all proteins in the profile being visualized as green signal while those detected specifically with the alkaline‐phosphatase conjugate appear as red signal. The dichromatic detection system is broadly compatible with a wide range of analytical imaging devices including UV epi‐ or transilluminators combined with photographic or charge‐coupled device (CCD) cameras, xenon‐arc sources equipped with appropriate excitation/emission filters, and dual laser gel scanners outfitted with a 473 nm second‐harmonic generation or 488 nm argon‐ion laser as well as a 633 nm helium‐neon or 635 nm diode laser. The dichromatic detection method permits detection of low nanogram amounts of protein and allows for unambiguous identification of target proteins relative to the entire protein profile on a single electroblot, obviating the need to run replicate gels that would otherwise require visualization of total proteins by silver staining and subsequent alignment with chemiluminescent or colorimetric signals generated on electroblots.

Optimization strategies for a fluorescent dye with bimodal excitation spectra: application to semiautomated proteomics

Facilities engaged in proteome analysis differ significantly in the degree that they implement automated systems for high-throughput protein characterization. Though automated workstation environments are becoming more routine in the biotechnology and pharmaceutical sectors of industry, university-based laboratories often perform these tasks manually, submitting protein spots excised from polyacrylamide gels to institutional core facilities for identification. For broad compatibility with imaging platforms, an optimized fluorescent dye developed for proteomics applications should be designed taking into account that laser scanners use visible light excitation and that charge-coupled device camera systems and gas discharge transilluminators rely upon UV excitation. The luminescent ruthenium metal complex, SYPRO Ruby protein gel stain, is compatible with a variety of excitation sources since it displays intense UV (280 nm) and visible (470 nm) absorption maxima. Localization is achieved by noncovalent, electrostatic and hydrophobic binding of dye to proteins, with signal being detected at 610 nm. Since proteins are not covalently modified by the dye, compatibility with downstream microchemical characterization techniques such as matrix-assisted laser desorption/ionization-mass spectrometry is assured. Protocols have been devised for optimizing fluorophore intensity. SYPRO Ruby dye outperforms alternatives such as silver staining in terms of quantitative capabilities, compatibility with mass spectrometry and ease of integration into automated work environments.