Improved Ruthenium II tris (bathophenantroline disulfonate) staining and destaining protocol for a better signal‐to‐background ratio and improved baseline resolution

Lamanda, Andreas; Zahn, Alain; Röder, Daniel; Langen, Hanno

doi:10.1002/pmic.200300587

Cited by 128 publications

(131 citation statements)

References 12 publications

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“…Generally, salicylate 1-hydroxylases have broad substrate specificity and can transform various monosubstituted salicylates (3,27,64). Unfortunately, no thorough investigation on kinetic parameters is available, and generally, only V max values or k cat values are given.…”

Section: Discussionmentioning

confidence: 99%

A Gene Cluster Involved in Degradation of Substituted Salicylates viaorthoCleavage inPseudomonassp. Strain MT1 Encodes Enzymes Specifically Adapted for Transformation of 4-Methylcatechol and 3-Methylmuconate

et al. 2007

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Pseudomonas sp. strain MT1 has recently been reported to degrade 4-and 5-chlorosalicylate by a pathway assumed to consist of a patchwork of reactions comprising enzymes of the 3-oxoadipate pathway. Genes encoding the initial steps in the degradation of salicylate and substituted derivatives were now localized and sequenced. One of the gene clusters characterized (sal) showed a novel gene arrangement, with salA, encoding a salicylate 1-hydroxylase, being clustered with salCD genes, encoding muconate cycloisomerase and catechol 1,2-dioxygenase, respectively, and was expressed during growth on salicylate and chlorosalicylate. A second gene cluster (cat), exhibiting the typical catRBCA arrangement of genes of the catechol branch of the 3-oxoadipate pathway in Pseudomonas strains, was expressed during growth on salicylate. Despite their high sequence similarities with isoenzymes encoded by the cat gene cluster, the catechol 1,2-dioxygenase and muconate cycloisomerase encoded by the sal cluster showed unusual kinetic properties. Enzymes were adapted for turnover of 4-chlorocatechol and 3-chloromuconate; however, 4-methylcatechol and 3-methylmuconate were identified as the preferred substrates. Investigation of the substrate spectrum identified 4-and 5-methylsalicylate as growth substrates, which were effectively converted by enzymes of the sal cluster into 4-methylmuconolactone, followed by isomerization to 3-methylmuconolactone. The function of the sal gene cluster is therefore to channel both chlorosubstituted and methylsubstituted salicylates into a catechol ortho cleavage pathway, followed by dismantling of the formed substituted muconolactones through specific pathways.

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Section: Discussionmentioning

confidence: 99%

A Gene Cluster Involved in Degradation of Substituted Salicylates viaorthoCleavage inPseudomonassp. Strain MT1 Encodes Enzymes Specifically Adapted for Transformation of 4-Methylcatechol and 3-Methylmuconate

et al. 2007

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“…Figure 3b shows the graph of the fluorescence intensity against protein amount of the minor protein present in the mix, b-galactosidase, ranging from 2799 to 16 pg. The new ruthenium stain allows protein visualization at the level of 80 pg with a good confidence, and allows detection of as few as 50 pg of proteins, a quantity that is not detected with SYPRO Ruby [23]. This last consideration is very important because the challenge in the protein detection field is to gain sensitivity in low protein quantities.…”

Section: Stain Performance Characteristics On 1-dementioning

confidence: 99%

High-sensitivity staining of proteins for one- and two-dimensional gel electrophoresis using post migration covalent staining with a ruthenium fluorophore

et al. 2006

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High-sensitivity staining of proteins for oneand two-dimensional gel electrophoresis using post migration covalent staining with a ruthenium fluorophore This paper describes the use of a ruthenium complex ((bis(2,2'-bipyridine)-4'-methyl-4-carboxybipyridine-ruthenium-N-succidimyl ester-bis(hexafluorophosphate), abbreviated below as ASCQ_Ru) commercially available and chemically pure. This new ruthenium complex ASCQ_Ru brings an activated ester, allowing the selective acylation of amino acid side chain amines for the post migration staining of proteins separated in 1-DE and 2-DE. The protocol used is a simple three-step protocol fixing the proteins in the gel, staining and then washing, as no lengthy destaining step is required. First the critical staining step was optimized. Although in solution the best described pH for acylating proteins with this reagent is phosphate buffer at pH 7.0, we found that best medium for in-gel staining is unbuffered ACN/water solution (20/80 v/v). The two other steps are less critical and classical conditions are satisfactory: fixing with 7% acetic acid/10% ethanol solution and washing four times for 10 min with water. Sensitivity tests were performed using 1-DE on protein molecular weight markers. We obtained a higher sensitivity than SYPRO ® Ruby with a detection limit of 80 pg of protein per well. However, contrary to SYPRO Ruby, ASCQ_Ru exhibits a logarithmic dependency on the amount of protein. The dynamic range is similar to SYPRO Ruby and is estimated between three and four orders of magnitude. Finally, the efficiency of the post migration ASCQ_Ru staining for 2-D gel separation is demonstrated on the whole protein extract from human colon carcinoma cells lines HCT 116. ASCQ_Ru gave the highest number of spot detected compared to other common stains Colloidal CBB, SYPRO Ruby and Deep Purple ™ .

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“…Following the first and second dimension, the gel was stained in ruthenium bathophenanthroline disulfonate (RuBPS) with an improved staining and destaining method (30). The spot picking, destaining, protein digestion, extraction, sample preparation, and spotting on matrix-assisted laser desorption ionization (MALDI) target plates were carried out using a spot handling work station (Ettan Spot Handling Work station, GE Healthcare) and a standard protocol provided by GE Healthcare.…”

Section: D-dige Protein Identificationmentioning

confidence: 99%

Impact of Temperature Dependent Sampling Procedures in Proteomics and Peptidomics – A Characterization of the Liver and Pancreas Post Mortem Degradome

Scholz

Sköld

Kultima

et al. 2011

Molecular & Cellular Proteomics

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Little is known about the nature of post mortem degradation of proteins and peptides on a global level, the socalled degradome. This is especially true for nonneural tissues. Degradome properties in relation to sampling procedures on different tissues are of great importance for the studies of, for instance, post translational modifications and/or the establishment of clinical biobanks. Here, snap freezing of fresh (<2 min post mortem time) mouse liver and pancreas tissue is compared with rapid heat stabilization with regard to effects on the proteome (using two-dimensional differential in-gel electrophoresis) and peptidome (using label free liquid chromatography). We report several proteins and peptides that exhibit heightened degradation sensitivity, for instance superoxide dismutase in liver, and peptidyl-prolyl cis-trans isomerase and insulin C-peptides in pancreas. Tissue sampling based on snap freezing produces a greater amount of degradation products and lower levels of endogenous peptides than rapid heat stabilization. We also demonstrate that solely snap freezing related degradation can be attenuated by subsequent heat stabilization.

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Improved Ruthenium II tris (bathophenantroline disulfonate) staining and destaining protocol for a better signal‐to‐background ratio and improved baseline resolution

Cited by 128 publications

References 12 publications

A Gene Cluster Involved in Degradation of Substituted Salicylates viaorthoCleavage inPseudomonassp. Strain MT1 Encodes Enzymes Specifically Adapted for Transformation of 4-Methylcatechol and 3-Methylmuconate

A Gene Cluster Involved in Degradation of Substituted Salicylates viaorthoCleavage inPseudomonassp. Strain MT1 Encodes Enzymes Specifically Adapted for Transformation of 4-Methylcatechol and 3-Methylmuconate

High-sensitivity staining of proteins for one- and two-dimensional gel electrophoresis using post migration covalent staining with a ruthenium fluorophore

Impact of Temperature Dependent Sampling Procedures in Proteomics and Peptidomics – A Characterization of the Liver and Pancreas Post Mortem Degradome

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