We report a simple gram scale synthesis of several imidazolinium salts (Br, Cl, PF6 and BF4), in this case 1,3–bis–(2,4,6–trimethylphenyl)imidazolinium bromide or chloride

Authors: Arnaud Gautier et al
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We report a simple gram scale synthesis of 1,3–bis–(2,4,6–trimethylphenyl)imidazolinium tetrafluoroborate or hexafluorophosphate

Authors: Arnaud Gautier et al
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Enolase (EC 4.2.1.11) is the second to last step in glycolysis and the third step in gluconeogenesis, catalyzing the reversible conversation of 2-phosphoglycerate and phosphoenolpyruvate. Enolase is a key enzyme in energy metabolism and measuring its enzymatic activity is of interest to investigators in diverse fields, including those studying cancer cell metabolism. We describe a simple and rapid...

Authors: Florian Muller, Elisa Quilanti and Ronald DePhinho
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This protocol describes a method to prepare detergent-solubilized membranes from Escherichia coli (E. coli), e.g. containing an overexpressed membrane protein. The procedure takes less than one day. Cells are broken by pressure cell and membranes are isolated and washed by differential centrifugation. Finally, the membranes are solubilized with the detergent of choice.

Authors: Daniel Harder and Dimitrios Fotiadis
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This protocol describes a method to purify a His-tagged membrane protein form detergent-solubilized Escherichia coli (E. coli) membranes. Solubilized membranes are incubated with Ni-NTA that binds the His-tagged protein. After washing, His-tagged proteins are eluted with histidine. When starting with solubilized membranes or membrane pellets before detergent solubilization, protein purification ta...

Authors: Daniel Harder and Dimitrios Fotiadis
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Here, we describe a protocol in which single cells are isolated acutely from adult mouse brains, made into single cell suspensions, depleted of myelin debris, then stained for markers to determine microglial contents. Cells are fixed and read on a multicolor flow cytometer.

Authors: Noel Dernecki, James Cronk and Jonathan Kipnis
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We have established an ultrafiltration-based in vitro assay for determining polyamine binding to proteins. In the protocol described here we have used Saccharomyces cerevisiae ODC antizyme protein produced in E. coli and radioactive polyamines to measure the direct binding of polyamines to ODC antizyme protein.

Authors: R Palanimurugan and R Jürgen Dohmen
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A new assay for the measurement of helicase enzyme activity was developed for the evaluation of the potency of potential inhibitors. This assay involves the use of a DNA or RNA duplex substrate and recombinant purified helicase. The DNA duplex consists of a pair of oligonucleotides, one of which is biotinylated and the other is digoxygenin (DIG)-labelled, both at their respective 5’ termini. Thi...

Authors: Dimitrios Vlachakis et al
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In dogs, a parasite burden is evident throughout the gastrointestinal tract but not is observed pathological alterations. Our aim was isolating lamina propria cells from dogs infected with leishmaniasis to further investigate if the parasite in the gut is correlated with the pathogenesis of the visceral infection. The biopsies were obtained of dogs and incubated with collagenase II. In the cells s...

Authors: Maria Marta Figueiredo et al
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This protocol describes a method developed in our laboratory to analyze repetitive DNA in chromosomes using fluorescence in situ hybridization (FISH) and flow cytometry (chromosome flow FISH or CFF). Our protocol includes details on procedures to isolate mitotic chromosomes from adherent cell cultures, FISH using peptide nucleic acid (PNA) probes on chromosomes in suspension and flow cytometry set...

Authors: Julie Brind’Amour and Peter M Lansdorp
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