Silver staining of proteins in polyacrylamide gels

Chevallet, Mireille; Luche, Sylvie; Rabilloud, Thierry

doi:10.1038/nprot.2006.288

Cited by 467 publications

(298 citation statements)

References 16 publications

Supporting

Mentioning

292

Contrasting

Unclassified

Order By: Relevance

“…Colloidal Coomassie staining of protein gels was carried out according to Neuhoff et al (1988). For a more sensitive detection of muscle proteins, silver staining was carried out by the method of Chevallet et al (2006). For image analysis, 6 gels representing young adult muscle and 6 gels representing aged muscle were scanned using Image Scanner II from GE Healthcare.…”

Section: Gel Electrophoretic Analysismentioning

confidence: 99%

Drastic increase of myosin light chain MLC-2 in senescent skeletal muscle indicates fast-to-slow fibre transition in sarcopenia of old age

Gannon

Doran

Kirwan

et al. 2009

European Journal of Cell Biology

View full text Add to dashboard Cite

The age-dependent decline in skeletal muscle mass and function is believed to be due to a multi-factorial pathology and represents a major factor that blocks healthy aging by increasing physical disability, frailty and loss of independence in the elderly. This study has focused on the comparative proteomic analysis of contractile elements and revealed that the most striking age-related changes seem to occur in the protein family representing myosin light chains (MLCs). Comparative screening of total muscle extracts suggests a fast-to-slow transition in the aged MLC population. The mass spectrometric analysis of the myofibril-enriched fraction identified the MLC2 isoform of the slow-type MLC as the contractile protein with the most drastically changed expression during aging. Immunoblotting confirmed an increased abundance of slow MLC2, concomitant with a switch in fast versus slow myosin heavy chains. Staining of two-dimensional gels of crude extracts with the phospho-specific fluorescent dye ProQ-Diamond identified the increased MLC2 spot as a muscle protein with a drastically enhanced phosphorylation level in aged fibres. Comparative immunofluorescence microscopy, using antibodies to fast and slow myosin isoforms, confirmed a fast-to-slow transformation process during muscle aging. Interestingly, the dramatic increase in slow MLC2 expression was restricted to individual senescent fibres. These findings agree with the idea that aged skeletal muscles undergo a shift to more aerobic-oxidative metabolism in a slower-twitching fibre population and suggest the slow MLC2 isoform as a potential biomarker for fibre type shifting in sarcopenia of old age.

show abstract

Section: Gel Electrophoretic Analysismentioning

confidence: 99%

Drastic increase of myosin light chain MLC-2 in senescent skeletal muscle indicates fast-to-slow fibre transition in sarcopenia of old age

Gannon

Doran

Kirwan

et al. 2009

European Journal of Cell Biology

View full text Add to dashboard Cite

show abstract

“…CBB-based stainings are simple to use and compatible with MS with a detection limit of around 8-100 ng/spot or 1-100 ng/spot for blue silver [101]. Silver staining is more sensitive, ,1-10 ng/spot but has a low reproducibility, a more complex time-consuming protocol and the most sensitive methods are not MS compatible [99,102].…”

Section: Protein Visualizationmentioning

confidence: 99%

Two‐dimensional gel electrophoresis and mass spectrometry for biomarker discovery

Penque

2009

Proteomics Clinical Apps

View full text Add to dashboard Cite

The proteome project, initiated in 1995, was made possible by 2-DE combined with MS. The project main objective was and remains the identification of all proteins expressed by a cell, tissue or organism in a given time and condition. Following this objective, the global profiling of proteins in health versus pathological state by the 2-DE/MS-based proteomic approach has contributed to the elucidation of the basic mechanisms of disease by discovering candidate disease biomarkers and disease targets for new drug development. This review will briefly summarize the historical evolution of 2-DE up to today, and review 2-DE/MS technology and its specific methods of study of immunoresponse (immunoproteomics), PTM of proteins, complex proteinprotein interactions (interactome), the proteome of cell membrane and intracellular proteome turnover in disease biomarker discovery. 2-DE historical evolutionGlobal profiling of proteins in healthy versus pathological states is a strategy to discover biomarkers for early diagnostics, disease progression, treatment response and novel targets for therapeutic drugs development. The idea of making an inventory of all the proteins in a cell, tissue or organism with normal or abnormal physiology, was (re)initiated in the middle of the 1990s with the proteome project [1]. Since then, many technologies to make the analysis of the proteome a reality have been united.The perfect technological 'marriage' that made the proteome project feasible was between high resolution 2-DE for separation of large numbers of proteins and MS for identification and characterization of the proteins displayed on this gel [2]. However, before this happy union occured, science and technology devoted to protein study had a long journey until 2-DE and MS combination was possible (Fig. 1). It started in the last century, in 1937, when Tiselius [3] introduced electrophoresis technique as a modification of Reiner's early concept (1927) [4] to analyse a complex mixture of proteins such as serum proteins. Only 20 years later, in 1959, electrophoresis on a gel support formed by crosslinked polymerization of Cyanogum 41, a commercial name for two organic monomers, acrylamide and the crosslinking agent, N,N1-methylenebisacrylamide, was shown to be useful and was named PAGE by Raymond and Weintraub [5]. The adaptation of polyacrylamide gel to IEF in a stable pH gradient, developed by Svensson in 1962 [6], was possible by the end of the 1960s [7][8][9]. About this time, the 2-D by combining IEF to separate undenatured proteins according to their charge and gradient SDS-PAGE for a second separation, according to their molecular mass, was for the first time introduced by Kenrick and Margolis (1970) [10]. Only in 1975, was IEF in denaturing conditions, as known today, used to follow the 2-D PAGE and it was described in three independent works [11][12][13]. The most powerful demonstraCorrespondence: Dr. Deborah Penque, Laboratório de Proteó-mica, Departamento de Genética, Instituto Nacional de Saúde, Dr. Ricardo Jorge, I.P., A...

show abstract

“…After that strips were placed on the top of 12% SDS polyacrylamide gels. The gels were run in the second dimension in Protean Plus TM Dodeca Cell TM electrophoretic chamber (Bio-Rad) at 40V for 1 h and then at 120V for 15 h at 10 o C. After 2-DE separation, analytical gels were visualized with silver stain (Chevallet et al 2006) and preparative gels with colloidal Coomassie Brilliant Blue G-250 (Candiano et al 2004). …”

Section: Two-dimensional Electrophoresis (2-de)mentioning

confidence: 99%

Blood plasma protein and lipid profile changes in calves during the first week of life

Herosimczyk¹,

Lepczyński²,

Ożgo³

et al. 2013

Polish Journal of Veterinary Sciences

View full text Add to dashboard Cite

The present study was undertaken to determine blood plasma protein and lipid profile changes in healthy Polish Holstein-Fresian calves of Black-and-White variety. Blood was drawn immediately after birth, before first colostrum intake and at the 3 Subsequent four blood samples were collected at 24 hour intervals until the 7 th day of life. Plasma proteins within the isoelectric point ranging from 3.0 to 10.0 were separated using high resolution two-dimensional electrophoresis. Among the 74 protein spots detected and analyzed, 16 were significantly altered during the first week of life. Differentially expressed spots were excised from the gels and subjected to peptide mass fingerprinting using MALDI-TOF MS. In total, 12 spots were successfully identified, which correspond to three proteins, namely: apolipoprotein A-I, apolipoprotein A-IV and fibrinogen gamma-B chain. A gradual increase in plasma triglyceride, total cholesterol, HDL and LDL cholesterol values was shown during the first seven days of calves life. The lowest concentration of these indicators were observed at birth and was followed by a rapid increase during the first week of postnatal life. These changes appear to be related to the transition in energy sources, from a maternal nutrient supply comprising mainly carbohydrates and amino acids to a diet which was rich in fat -colostrum and milk. This was reflected by the intense up-regulation of plasma proteins related with lipid transport and lipoprotein metabolism during the first week of life.

show abstract

Silver staining of proteins in polyacrylamide gels

Cited by 467 publications

References 16 publications

Drastic increase of myosin light chain MLC-2 in senescent skeletal muscle indicates fast-to-slow fibre transition in sarcopenia of old age

Drastic increase of myosin light chain MLC-2 in senescent skeletal muscle indicates fast-to-slow fibre transition in sarcopenia of old age

Two‐dimensional gel electrophoresis and mass spectrometry for biomarker discovery

Blood plasma protein and lipid profile changes in calves during the first week of life

Contact Info

Product

Resources

About