scholarly journals Major changes in phosphorylation of chromatin-associated non-histone proteins accompany development in the slime mould Dictyostelium discoideum

1985 ◽  
Vol 229 (3) ◽  
pp. 771-778 ◽  
Author(s):  
J H Sinclair ◽  
D Rickwood
Keyword(s):  
Dictyostelium Discoideum ◽  
Blue Staining ◽  
Two Dimensional Gel Electrophoresis ◽  
Transcription Analysis ◽  
Slime Mould ◽  
Coomassie Blue Staining ◽  
Cytoplasmic Rna ◽  
Coomassie Blue ◽  
Protein And Rna Synthesis ◽  
Development Phases

Striking changes in protein and RNA synthesis were shown to accompany development in the slime mould Dictyostelium discoideum. These changes are, at least in part, directly attributable to control at the level of transcription. Analysis of nuclear proteins and their states of phosphorylation by two-dimensional gel electrophoresis and autoradiography showed only minor changes in the species of proteins detectable by Coomassie Blue staining during the vegetative growth and development phases. Major changes, however, were detected in their patterns of phosphorylation, with major differences from the vegetative pattern occurring during both early development (0-2h) and late development (8-12h). These changes coincide with major changes in polypeptide synthesis and in nuclear and cytoplasmic RNA complexity.

scholarly journals The effect of cultureware surfaces on functional and structural components of differentiated 3T3-L1 preadipocytes

2015 ◽  
Vol 20 (5) ◽  
Author(s):  
Nela Pavlikova ◽  
Martin Weiszenstein ◽  
Jan Pala ◽  
Petr Halada ◽  
Ondrej Seda ◽  
...  
Keyword(s):  
Pathway Analysis ◽  
Cultured Cells ◽  
Culture Conditions ◽  
Blue Staining ◽  
Ionization Mass ◽  
Two Dimensional Gel Electrophoresis ◽  
Coomassie Blue Staining ◽  
Coomassie Blue ◽  
The Impact

AbstractExperiments using cultured primary cells or cell lines are a routine in vitro approach used across multiple biological disciplines, However, the structural and functional influences of various cultureware materials on cultured cells is not clearly understood. Surface treatments of cultureware have proven to have profound effects on cell viability and proliferation. In this study, we investigated the impact of polystyrene and fluorocarbon cultureware dishes on the proteomic profile of differentiated 3T3-L1 preadipocytes. After expansion and differentiation of cells on appropriate cultureware dishes, cell lysates were separated using two-dimensional gel electrophoresis and proteins were visualized with Coomassie blue staining. Spots with the highest differential expression between the two culture conditions were subsequently analyzed using matrix-assisted laser desorption/ionization mass spectrometry and the identified proteins were subjected to pathway analysis. We observed that 43% of all spots were differentially expressed depending on the cultureware. Pathway analysis revealed that glucose metabolism, mitochondrial structure and cell differentiation, represented by 14-3-3 protein-mediated signaling and the mitochondrial inner membrane organizing system (MINOS), were significantly affected by cultureware material. These results indicate that cultureware material can have a profound effect on key adipocyte functional pathways. These effects modifications of the cells should be reflected in the design of in vitro experiments and interpretation of their results.

Proteomic analysis ofEntamoeba histolytica

Parasitology ◽  
2006 ◽  
Vol 134 (2) ◽  
pp. 289-298 ◽  
Author(s):  
J. TOLSTRUP ◽  
E. KRAUSE ◽  
E. TANNICH ◽  
I. BRUCHHAUS
Keyword(s):  
Ubiquitin Proteasome System ◽  
Blue Staining ◽  
Two Dimensional Gel Electrophoresis ◽  
Coomassie Blue Staining ◽  
Coomassie Blue ◽  
Peptide Mass ◽  
Resultant Data ◽  
Ubiquitin Proteasome ◽  
Ph Range ◽  
First Time

In this study, the proteome of axenically grownEntamoeba histolyticaparasites was explored by two-dimensional gel electrophoresis (2-DE), employing a practical and effective procedure for the solubilization ofE. histolyticaproteins. Approximately 900 protein species in the pH range between 4 and 7 were detected by Coomassie Blue staining. Ninety-five spots were excised, trypsinated and subjected to mass spectrometry. The resultant data from peptide mass fingerprints were compared with those available in theE. histolyticagenome and the (non-redundant) National Center for Biotechnology Information (NCBI) databases for the identification and categorization of proteins. Sixty-three of the proteins identified were predicted to relate to the cytoskeleton, surface, glycolysis, RNA/DNA metabolism, the ubiquitin-proteasome system, vesicular trafficking and signal transduction. The present study demonstrates, for the first time, that corresponding genes are indeed expressed inE. histolyticaand provides a foundation for further proteomic studies of this parasite.

Coomassie blue staining for high sensitivity gel-based proteomics

2013 ◽  
Vol 90 ◽  
pp. 96-106 ◽  
Author(s):  
Victoria J. Gauci ◽  
Matthew P. Padula ◽  
Jens R. Coorssen
Keyword(s):  
High Sensitivity ◽  
Blue Staining ◽  
Coomassie Blue Staining ◽  
Coomassie Blue

scholarly journals Heating Greatly Speeds Coomassie Blue Staining and Destaining

BioTechniques ◽  
2000 ◽  
Vol 28 (3) ◽  
pp. 426-432 ◽  
Author(s):  
Connie Wong ◽  
Srinavas Sridhara ◽  
James C.A. Bardwell ◽  
Ursula Jakob
Keyword(s):  
Blue Staining ◽  
Coomassie Blue Staining ◽  
Coomassie Blue

The reactivity to N-ethyl maleimide of the subunits of cytochrome oxidase

1977 ◽  
Vol 55 (9) ◽  
pp. 988-994 ◽  
Author(s):  
A. McGeer ◽  
B. Lavers ◽  
G. R. Williams
Keyword(s):  
Electron Transport ◽  
Cytochrome Oxidase ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Blue Staining ◽  
Beef Heart ◽  
Coomassie Blue Staining ◽  
Coomassie Blue

Beef heart cytochrome oxidase (EC 1.9.3.1) prepared in this laboratory consistently presents 10 Coomassie blue staining zones on SDS–polyacrylamide gel electrophoresis. At pH 7.0 only two of these polypeptides (III and VIa) are labelled by radioactive N-ethyl maleimide (NEM). The labelling of VIa is variable and correlates with the activity of particular oxidase preparations. When cytochrome oxidase is isolated from alkylated membranes, either mitochondria or electron transport particles, polypeptide VIa is found not to be labelled; polypeptide III is more strongly labelled than when isolated oxidase is alkylated, and label now appears in polypeptide I which is not alkylated upon treatment of isolated oxidase with NEM.

PRODUCTION AND CHARACTERISATION OF A MONOCLONAL ANTIBODY AGAINST HUMAN ANTI-THROMBIN III

1987 ◽  
Author(s):  
Deborah A Rathjen ◽  
Carolyn L Geczy
Keyword(s):  
Cultured Cells ◽  
Factor Xa ◽  
Lymphocyte Activation ◽  
Blue Staining ◽  
Aortic Endothelial Cells ◽  
Tissue Sections ◽  
Coomassie Blue Staining ◽  
Coomassie Blue ◽  
At Iii

To study the role of anticoagulants, particularly antithrombin III (AT III) and heparin, on the activation of coagulation by monocytes/macrophages which have been stimulated with a soluble lymphocyte activation product, macrophage procoagulant inducing factor, we have prepared monoclonal antibodies (MAbs) to human AT III.In fusion experiments, in contrast to wells containing peritoneal feeder cells, positive hybrids were only found in wells containing medium conditioned by the macrophage cell line J774 (Rathjen and Geczy, 1986). Of 5 hybrids which initially produced antibody, only one hybrid, showed stable Ab production. The MAb, designated 22/23, was not cross-reactive with either 1 antitrypsin or ovalbumin and did not inhibit the biological activity of AT III in chromogenic assays which measured inhibition of thrombin and Factor Xa by AT III. An immunoadsorbent prepared using MAb 22/23 depleted AT III activity from a purified AT III preparation. Reduction and alkylat ion of the disulphide bonds of the protein portion of AT III completely abbrogated MAb binding indicating that the native configuration of AT III was important. Isoelectric focussing of AT III, followed by transfer of the focussed protein to nitrocellulose by diffusion and probing with MAb 22/23, revealed at least 8 bands in the region of pH 5.2 to 5.85. Coomassie blue staining of a gel run in parallel showed 9 bands in this region. The MAb provides a useful tool for the detection of AT III on both cultured cells (bovine aortic endothelial cells are positive by immunofluorescence) and tissue sections.Rathjen, D.A. and Geczy, C.L. Hybridomo. 5s 255-261 (1986)

scholarly journals Immunochemical characterization of the platelet-specific alloantigen Leka: a comparative study with the PlA1 alloantigen

Blood ◽  
1984 ◽  
Vol 64 (6) ◽  
pp. 1212-1219 ◽  
Author(s):  
N Kieffer ◽  
B Boizard ◽  
D Didry ◽  
JL Wautier ◽  
AT Nurden
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Human Platelets ◽  
Blue Staining ◽  
Igg Antibody ◽  
Immune Disorder ◽  
Immunochemical Characterization ◽  
Coomassie Blue Staining ◽  
Coomassie Blue

Abstract We report the immunochemical characterization of a new platelet- specific alloantigen detected using an IgG antibody isolated from the serum of a patient with posttransfusion purpura (PTP). In indirect immunoprecipitation experiments, the antibody, termed anti-Leka, predominantly precipitated glycoprotein (GP) IIb from Triton X-100 lysates of normal human platelets. In an immunoblot procedure, which involved the transfer of platelet polypeptides separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to nitrocellulose membrane, anti-Leka bound exclusively to GP IIb. Under identical conditions, four anti-PlA1 antibodies each reacted with GP IIIa. No binding of anti-Leka IgG occurred to Leka (-) platelets or to their separated polypeptides although GP IIb was normally detected by Coomassie blue staining. After electrophoresis of reduced platelet proteins, the Leka determinant was localized to the IIb alpha chain. Thus, unlike the PlA1 antigen, the Leka determinant was not destroyed by disulfide reduction. Analysis of platelets from a patient with Glanzmann's thrombasthenia revealed little or no binding in the GP IIb position. Anti-Leka permitted the identification of 76,000 and 60,000 dalton fragments of GP IIb retained by the platelet following chymotrypsin treatment. Our results further highlight the immunogenicity of the GP IIb-IIIa complex. They also suggest that antibodies against GP IIb can cause the thrombocytopenia observed in PTP and that anti-PlA1 antibodies do not account exclusively for the pathophysiology of this immune disorder.

Oviductal fluid protein patterns in the rhesus monkey (Macaca mulatta) during the menstrual cycle

1992 ◽  
Vol 4 (2) ◽  
pp. 249 ◽  
Author(s):  
A Paliwal ◽  
B Malaviya ◽  
VP Kamboj
Keyword(s):  
Menstrual Cycle ◽  
Protein Profile ◽  
Periodic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Blue Staining ◽  
Protein Patterns ◽  
Coomassie Blue Staining ◽  
Coomassie Blue ◽  
Oviductal Fluid

Oviducts were obtained from monkeys on Days 8, 14, 19 and 25 of the menstrual cycle and changes in the pattern of luminal fluid proteins were examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Densitometric analysis after periodic acid Schiff's reagent (PAS) and coomassie blue staining of the gels revealed 85 and 95 kDa proteins only up to Day 14 whereas a 130 kDa glycoprotein persisted up to Day 19 and reached a nadir at mid-menstrual cycle (Day 14). The absence of the 130 kDa glycoprotein in the serum and its presence in cytosolic preparations up to Day 19 suggest that it is of oviductal origin. The 130 kDa glycoprotein is of particular interest since it was present in the oviductal fluid during mid cycle, a period when the oviduct participates in gamete transport, fertilization and embryo development. The conclusion drawn from this study is that the protein profile of monkey oviductal fluid changes during the menstrual cycle.

scholarly journals Detection in milk of a 60,000 Mr mouse and a 68,000 Mr human phosphorylated glycoprotein by histochemical analysis on polyacrylamide gels.

1983 ◽  
Vol 31 (6) ◽  
pp. 709-716 ◽  
Author(s):  
M R Green
Keyword(s):  
Molecular Weight ◽  
Sialic Acid ◽  
Periodic Acid ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Blue Staining ◽  
Polyacrylamide Gels ◽  
Periodic Acid Schiff ◽  
Coomassie Blue Staining ◽  
Coomassie Blue

Proteins in colostrum and skimmed milk from humans and mice were separated by electrophoresis on polyacrylamide gels and stained with Coomassie blue (CB), Ethyl-Stains-all (ESA), and periodic acid-Schiff (PAS) to investigate changes that may occur in milks throughout lactation. In mouse colostrum but not in mature mouse milk, a PAS-positive protein of apparent molecular weight of 60,000 stained prominently blue with ESA. A protein in human milk with a molecular weight of 68,000 stained similarly but was present throughout lactation. The intensity of blue staining of these minor proteins in milk approached that obtained with casein phosphoproteins. The metachromatic dye ESA stains phosphoproteins and sialic acid-rich glycoproteins blue to blue-green. Removal of phosphorus from the former and sialic acid from the latter results in those proteins staining red with ESA. The intensity of blue staining of the 60,000 and 68,000 Mr proteins was diminished but not lost following treatment with phosphatase. It was eliminated following neuraminidase digestion of the mouse protein and mild acid hydrolysis of the human protein. Coomassie blue staining of the proteins was not affected by these procedures. Following electrophoresis of milk and milk fractions in a non-sodium dodecyl sulfate-containing system, the proteins were identified by their characteristic staining properties with ESA and isolated.

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