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

Abstract

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

“…A range of fixatives were evaluated in this study including 40% ethanol/10% acetic acid, 10% ethanol/7% acetic acid, 25% ethanol/ 12.5% TCA and 10% ethanol/3% phosphoric acid. Minimal staining volumes were found to be 50 mL for 8 cm 6 10 cm60.75 mm minigels, or 500 mL for 20 cm6 20 cm61 mm large format 2-D gels [27]. This corresponds to a solution volume that is roughly 10 times the volume of the gel.…”

Sequential electroelution and mass spectroscopic identification of intact sodium dodecyl sulfate- proteins labeled with 5(6)-carboxyfluorescein-N-hydroxysuccinimide ester

“…In view of the present commercial unavailability of the HPGE apparatus and the nonexistence of any other apparatus for the intermittent scanning of a gel electrophoretic migration path with capability of computer-directed band retrieval, however, the post-staining of protein by SYPRO dyes (e.g., [7][8][9][10]) may appear as an alternative to prestaining with FLUOS for the purpose of band transfer into MS. As pointed out above, the reversibility of post-staining with fluorescent SYPRO red, orange, or ruby is due to their reactivity towards SDS, not protein, so that upon removal of SDS prior to MS these dyes are also removed and do not interfere with MS. A new, albeit still primitive, electroelution apparatus [22] lends itself to post-staining, by contrast with the HPGE scanning apparatus, and therefore to the application of fluorescent staining with SYPRO dyes. The approach remains nonetheless problematic in view of the inapplicability of electroelution of intact proteins with fixation of protein bands, and the fact that without fixation, the sensitivity of SYPRO stains for SDS-proteins is low.…”

“…Failure to be susceptible to proteolysis apparently may be specific for FLUOS. Reaction of protein amino groups with 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) does not inhibit tryptic hydrolysis [8]. The causes of the discrepancy remain unknown and to be investigated.…”

“…An alternative way of reversible fluorescent labeling of SDS-proteins is that with SDS-reactive SYPRO stains [7][8][9][10]. Since these dyes react with SDS (presumably micellar SDS) which is complexed with the protein, not with the protein itself, they do not interfere with MS of the protein after removal of SDS.…”

High throughput approaches in neuroscience

“…Recently, fluorescent protein dyes such as SYPRO stains and CyDyes with similar sensitivity to silver stains have begun to be used. These newer fluorescent dyes are also more compatible with protein identification by mass spectrometry, other methods often requiring de‐staining before identification (Lauber et al, 2001, Berggren et al, 2002; Berggren et al, 2000; Berggren et al, 2001). Traditionally, one of the biggest disadvantages of 2D‐PAGE was poor reproducibility, however with the advent of fluorescence techniques, this has been greatly improved, though silver and Coomassie stains are still routinely used in many labs, and routinely used to generate proteome databases of various organisms or tissues (see http://ca.expasy.org/ch2d/).…”