Polyacrylamide gel staining with Fe2+-bathophenanthroline sulfonate

Graham, G.; Nairn, Rodney S.; Bates, George W.

doi:10.1016/0003-2697(78)90441-4

Cited by 21 publications

(20 citation statements)

References 11 publications

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“…First, EutT activity was lost upon exposure to oxygen. Second, EutT activity was lost as a function of the concentration of bathophenanthroline (an Fe 2ϩ chelator) (16) in the reaction mixture (Fig. 5).…”

Section: -ء‬mentioning

confidence: 99%

The eutT Gene of Salmonella enterica Encodes an Oxygen-Labile, Metal-Containing ATP:Corrinoid Adenosyltransferase Enzyme

2004

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The eutT gene of Salmonella enterica was cloned and overexpressed, and the function of its product was established in vivo and in vitro. The EutT protein has an oxygen-labile, metal-containing ATP:co(I)rrinoid adenosyltransferase activity associated with it. Functional redundancy between EutT and the housekeeping ATP: co(I)rrinoid adenosyltransferase CobA enzyme was demonstrated through phenotypic analyses of mutant strains. Lack of CobA and EutT blocked ethanolamine utilization. EutT was necessary and sufficient for growth of an S. enterica cobA eutT strain on ethanolamine as a carbon and energy or nitrogen source. A eutT ؉ gene provided in trans corrected the adenosylcobalamin-dependent transcription of a eut-lacZ operon fusion in a cobA strain. Cell extracts enriched for EutT protein contained strong, readily detectable ATP:co(I)rrinoid adenosyltransferase activity. The activity was only detected in extracts maintained under anoxic conditions, with complete loss of activity upon exposure to air or treatment with the Fe 2؉ ion chelator bathophenanthroline. While the involvement of another metal ion cannot be ruled out, the observed sensitivity to air and bathophenanthroline suggests involvement of Fe 2؉ . We propose that the EutT protein is a unique metal-containing ATP:co(I)rrinoid adenosyltransferase. It is unclear whether the metal ion plays a structural or catalytic role.The biosynthesis of AdoCbl (coenzyme B 12 ) is unique to some prokaryotes. Multiple functions (encoded by Ͼ25 genes) are required for the assembly of this complex molecule (36). The chief structural feature of AdoCbl is the presence of an adenosyl moiety liganded to the Co ion of cobalamin via a covalent Co-C bond. In the enterobacterium Salmonella enterica, corrinoid adenosylation is required for de novo synthesis and for the assimilation of incomplete precursors such as cobinamide (12). Insights into the corrinoid adenosylation pathway in S. enterica ( Fig. 1) were recently reported, including the three-dimensional crystal structure of the ATP:co(I)rrinoid adenosyltransferase (CobA) enzyme responsible for the formation of the Co-C bond (2,(13)(14)(15)35). The cobA gene in S. enterica is not part of the 17-gene cob operon (27, 33) and appears to be constitutively expressed (34). In this bacterium, CobA is the housekeeping adenosyltransferase needed for de novo biosynthesis and for the assimilation of exogenous corrinoids. However, CobA is not the only corrinoid adenosyltransferase present in S. enterica. Other large operons like the 1,2-propanediol utilization (pdu) and ethanolamine utilization (eut) operons appear to encode their own corrinoid adenosyltransferases. Johnson et al. recently reported evidence that the pduO gene of S. enterica encodes an ATP:cobalamin adenosyltransferase (19). The eutT gene of the eut operon was suggested to encode the corrinoid adenosyltransferase enzyme for this pathway, but support for this assignment was inconclusive (20). Interestingly, CobA, PduO, and EutT do not have an ancestor in common, suggesting ...

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Section: -ء‬mentioning

confidence: 99%

The eutT Gene of Salmonella enterica Encodes an Oxygen-Labile, Metal-Containing ATP:Corrinoid Adenosyltransferase Enzyme

2004

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“…Significantly, despite the high sensitivity of many silver staining methods, staining with silver occurs in a largely nonstoichiometric manner, has a relatively low linear range and suffers from considerable run-to-run variability. SYPRO Ruby is a recently introduced reagent based on a series of transition metal chelate stains first introduced over 20 years ago [10][11][12][13][14][15]. Ruthenium is chelated into a complex as a fluorophore and operates without the need for protein-modifying agents such as glutaraldehyde and formaldehyde, thus contributing to increased compatibility with mass spectrometric techniques.…”

Section: Introductionmentioning

confidence: 99%

A statistical comparison of silver and SYPRO Ruby staining for proteomic analysis

et al. 2004

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Silver staining has been the method most commonly employed for high sensitivity staining of proteins following two-dimensional gel electrophoresis. Whilst this method offers detection in the nanogram range it does have major drawbacks including a lack of linearity, nonstoichiometric staining of proteins, a lack of compatibility with the microchemical preparation of proteins for identification by mass spectrometric techniques, and a highly subjective assessment of the staining endpoint. SYPRO Ruby is a relatively new, ruthenium complex-based stain which is reported to offer advantages over silver, particularly in overcoming the limitations cited above. We describe a series of experiments where several protein staining procedures commonly employed are compared. To enable optimization of the in situ digestion procedure, a statistical approach has been undertaken. The effects of a variety of staining, digestion, and analysis protocols on the downstream processing of a test radiolabeled protein were studied. The data confirms that as well as offering sensitivity similar to silver, SYPRO Ruby staining is reproducible, linear, and offers a higher level of compatibility with the identification of proteins by mass spectrometry.

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“…In contrast to the direct-labeling approach, the indirect-labeling method can be adapted to detect any class of substances for which a highly neutron-activatable, selectively binding ligand is available. The theoretically achievable sensitivity of the indirect-labeling method is in the attomole (10)(11)(12)(13)(14)(15)(16)(17)(18) mol) range.…”

Section: Introductionmentioning

confidence: 99%

“…'51Eu and l64Dy are among the most highly activatable elements, with thermal neutron activation cross sections of 3000 and 2600 barns, respectively (l52mEu, t,12 9.3 hr; 165Dy, til2 2.3 hr). BPS (in a complex with multivalent metal ions) binds to proteins and has been proposed as a general protein stain (14).…”

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confidence: 99%

Post-separation detection of nucleic acids and proteins by neutron activation.

Snapka¹,

Kwok²,

Bernard³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

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We describe approaches to neutron activation analysis and their application to post-separation autoradiographic detection of biological compounds. Specifically, we have extended the use of a "direct-labeling" method to the post-separation detection of DNA after gel electrophoresis and to the detection of nucleotides separated by TLC. In addition, we describe a more generally applicable "indirect-labeling" method in which separated compounds of interest are selectively bound to ligands containing highly neutron-activatable elements, such as manganese (5 Mn), europium ('5'Eu), or dysprosium (164Dy), and then irradiated with thermal neutrons. This method is illustrated with nucleotides separated by TLC and with prc teins separated by polyacrylamide gel electrophoresis. In contrast to the direct-labeling approach, the indirect-labeling method can be adapted to detect any class of substances for which a highly neutron-activatable, selectively binding ligand is available. The theoretically achievable sensitivity of the indirect-labeling method is in the attomole (10-18 mol) range.Radioactive labeling is one of the most sensitive analytical strategies for the detection of chemical compounds. However, applications of this approach in biochemistry are often subject to significant constraints. In vivo radiolabeling may be complicated by radiation damage to cells and by competition from intracellular pools of precursors (1-5). In vitro radiolabeling, though producing much higher specific radioactivities, often results in undesired chemical modification of the original compounds, which may greatly complicate their separation and identification unless radiolabeling is carried out after separation of the original compounds. As suggested more than 30 years ago (6), one general method for such post-separation labeling is that of neutron activation. This technique is based upon selective conversion (by neutron capture) of some of the atoms of the sample molecules to unstable isotopes whose radioactive decay is readily detected.In the "direct-labeling" method of post-separation detection by means of neutron activation, one or more of the elements present in the separated compounds of interest are converted directly to their radioactive isotopes by irradiation with thermal neutrons in situ. In an early application of this approach, phosphorus (31P)-containing compounds, such as phospholipids, were detected after their separation by paper chromatography by using neutron activation to convert 31p into radioactive 32p (7)(8)(9)(10)(11)(12). In the present work we have extended this direct-labeling method to the detection of nucleotides separated by TLC and to the detection of DNA separated by agarose gel electrophoresis. Neutron activation of 31p in the above compounds yields detection sensitivity comparable to that obtained by in vivo labeling and has the advantage that molecular species to be detected are labeled only after their separation.In addition, we describe a more generally applicable "indirect-labeling" method of p...

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Polyacrylamide gel staining with Fe2+-bathophenanthroline sulfonate

Cited by 21 publications

References 11 publications

The eutT Gene of Salmonella enterica Encodes an Oxygen-Labile, Metal-Containing ATP:Corrinoid Adenosyltransferase Enzyme

The eutT Gene of Salmonella enterica Encodes an Oxygen-Labile, Metal-Containing ATP:Corrinoid Adenosyltransferase Enzyme

A statistical comparison of silver and SYPRO Ruby staining for proteomic analysis

Post-separation detection of nucleic acids and proteins by neutron activation.

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