Staining of alkali-labile phosphoproteins and alkaline phosphatases on polyacrylamide gels

Debruyne, Ignace

doi:10.1016/0003-2697(83)90229-4

Cited by 35 publications

(19 citation statements)

References 14 publications

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“…Because of its poor specificity and low sensitivity, "Stains-All" is not used to detect phosphoproteins. The Methyl Green dye [18,19], which detects phosphoproteins through the formation of an insoluble phosphomolybdate, is also not suitable for the detection of low abundance phosphoproteins because its staining ability is about 80-160 ng phosvitin for 100 phosphoserine residues. However, the Pro-Q Diamond dye that we selected has good specificity for phosphoserine-, phosphothreonine-, and phosphotyrosine-containing proteins and does not stain DNA, RNA, or sulfated glycans.…”

Section: Selection Of Staining Methodsmentioning

confidence: 99%

Phosphoproteome profile of human liver Chang's cell based on 2‐DE with fluorescence staining and MALDI‐TOF/TOF‐MS

et al. 2007

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Because reversible protein phosphorylation is central to biological regulation, many methods have been developed for the systematic parallel analysis of the phosphorylation status of large sets of proteins. To directly survey the extent of protein phosphorylation and the distribution of phosphoproteins in biological systems, we used a phosphoprotein staining method, Pro-Q Diamond dye, for the high-throughput identification of phosphoproteins. The specificity of the method was validated with protein standards and subsequently applied to an analysis of total protein from human liver Chang's cells. Proteins were separated by 2-DE, then sequentially stained with Pro-Q Diamond and Coomassie Blue G-250. After image analysis, the proteins in gel spots containing phosphoproteins were identified by MALDI-TOF/TOF-MS. A total of 269 phosphoproteins were identified, and 27 were known phosphoproteins in the SwissProt database. By comparing the relative volumes of the phosphoprotein map and the total protein map, the extent of protein phosphorylation was observed. The phosphoprotein staining method combined with 2-DE also detected polymorphisms of the phosphoproteins, and could distinguish highly abundant, but slightly phosphorylated proteins from less abundant, highly phosphorylated ones. We conclude that the phosphoprotein staining method can be used for global, quantitative phosphorylation detection.

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Section: Selection Of Staining Methodsmentioning

confidence: 99%

Phosphoproteome profile of human liver Chang's cell based on 2‐DE with fluorescence staining and MALDI‐TOF/TOF‐MS

et al. 2007

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“…Since 1970 several methods have been described [26,27] to stain phosphoproteins directly in gels but the low specificity and sensitivity prevented those methods from being routinely applied. In fact, in some cases such dyes did not specifically discriminate between phosphorylated and unphosphorylated proteins whereas in other cases only phosphoserine and phosphothreonine were detected.…”

Section: Direct Staining Of Phosphoproteinsmentioning

confidence: 99%

Phosphoproteome Analysis

2005

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Protein phosphorylation is directly or indirectly involved in all important cellular events. The understanding of its regulatory role requires the discovery of the proteins involved in these processes and how, where and when protein phosphorylation takes place. Investigation of the phosphoproteome of a cell is becoming feasible today although it still represents a very difficult task especially if quantitative comparisons have to be made. Several different experimental strategies can be employed to explore phosphoproteomes and this review will cover the most important ones such as incorporation of radiolabeled phosphate into proteins, application of specific antibodies against phosphorylated residues and direct staining of phosphorylated proteins in polyacrylamide gels. Moreover, methods to enrich phosphorylated proteins such as affinity chromatography (IMAC) and immunoprecipitation as well as mass spectrometry for identification of phosphorylated peptides and phosphorylation sites are also described.

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“…Methyl Green dye can also be used to visualize phosphoproteins in gels through the formation of an insoluble phosphomolybdate complex. Unfortunately, the detection sensitivity of Methyl Green is considerably poorer than that of Coomassie blue staining [18,19]. Pro-Q  Diamond phosphoprotein gel stain from Molecular Probes (Eugene, OR, USA) is a new fluorescence-based technique for detection of phosphoproteins.…”

Section: Introductionmentioning

confidence: 99%

Functional characterization of two-dimensional gel-separated proteins using sequential staining

Lenchik

Pabst

et al. 2005

Electrophoresis

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Proteins separated by two-dimensional (2-D) gel electrophoresis can be visualized using various protein staining methods. This is followed by downstream procedures, such as image analysis, gel spot cutting, protein digestion, and mass spectrometry (MS), to characterize protein expression profiles within cells, tissues, organisms, or body fluids. Characterizing specific post-translational modifications on proteins using MS of peptide fragments is difficult and labor-intensive. Recently, specific staining methods have been developed and merged into the 2-D gel platform so that not only general protein patterns but also patterns of phosphorylated and glycosylated proteins can be obtained. We used the new Pro-Q Diamond phosphoprotein dye technology for the fluorescent detection of phosphoproteins directly in 2-D gels of mouse leukocyte proteins, and Pro-Q Emerald 488 glycoprotein dye to detect glycoproteins. These two fluorescent stains are compatible with general protein stains, such as SYPRO Ruby stain. We devised a sequential procedure using Pro-Q Diamond (phosphoprotein), followed by Pro-Q Emerald 488 (glycoprotein), followed by SYPRO Ruby stain (general protein stain), and finally silver stain for total protein profile. This multiple staining of the proteins in a single gel provided parallel determination of protein expression and preliminary characterization of post-translational modifications of proteins in individual spots on 2-D gels. Although this method does not provide the same degree of certainty as traditional MS methods of characterizing post-translational modifications, it is much simpler, faster, and does not require sophisticated equipment and expertise in MS.

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Staining of alkali-labile phosphoproteins and alkaline phosphatases on polyacrylamide gels

Cited by 35 publications

References 14 publications

Phosphoproteome profile of human liver Chang's cell based on 2‐DE with fluorescence staining and MALDI‐TOF/TOF‐MS

Phosphoproteome profile of human liver Chang's cell based on 2‐DE with fluorescence staining and MALDI‐TOF/TOF‐MS

Phosphoproteome Analysis

Functional characterization of two-dimensional gel-separated proteins using sequential staining

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