RESUMO
Protein synthesis is an essential process that affects major cellular functions including growth, energy production, cell signaling, and enzymatic reactions. However, how it is impacted by aging and how the translation of specific proteins is changed during the aging process remain understudied. Although yeast is a widely used model for studying eukaryotic aging, analysis of age-related translational changes using ribosome profiling in this organism has been challenging due to the need for isolating large quantities of old cells. Here, we provide a detailed protocol for genome-wide analysis of protein synthesis using ribosome profiling in replicatively aged yeast. By combining genetic enrichment of old cells with the biotin affinity purification step, this method allows large-scale isolation of aged cells sufficient for generating ribosome profiling libraries. We also describe a strategy for normalization of samples using a spike-in with worm lysates that permits quantitative comparison of absolute translation levels between young and old cells.
Assuntos
Caenorhabditis elegans/química , RNA Mensageiro/genética , Ribossomos/metabolismo , Saccharomyces cerevisiae/fisiologia , Animais , Biotina/química , Meios de Cultura/química , Replicação do DNA , Sequenciamento de Nucleotídeos em Larga Escala , Biossíntese de Proteínas , RNA Mensageiro/química , Ribossomos/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Análise de Sequência de RNARESUMO
We report an inkjet-printed paper based colorimetric sensor with silver nanoparticles (AgNPs) using smartphone and color detector App for on-site determination of mercuric ion (Hg2+) from environmental water samples. The AgNPs printed on Whatman filter paper (No. 1) is employed for detection of Hg2+ which is reliant on the color change of NPs from yellow to discoloration depending on the concentration of target analyte in sample solution. The quantitative determination was performed by calculating the signal intensity of AgNPs on printed paper substrate after the introduction of Hg2+ using smartphone and RGB color detector. The mechanism for detection of Hg2+ on paper substrate is verified using UV-Vis spectrophotometry (UV-Vis), transmission electron microscope (TEM), X-ray photoelectron spectroscopy (XPS), dynamic light scattering (DLS) and basic chemical assays. The linear range acquired for paper based colorimetric detection in the range of 40-1200 µgL-1 with limit of detection of 10 µgL-1. The results obtained using an inkjet-printed paper-based chemical sensor combined with a smartphone is validated with data of inductively coupled plasma-atomic emission spectroscopy (ICP-AES) measurement. The advantages of paper based detection are simple, rapid, economic and can be applied at the sample sources for determination of Hg2+.
RESUMO
The results of a study and application of leucocrystal violet for the determination of antimony in parts per million levels is described here. The proposed method is based on the reaction of antimony(III) with acidified potassium iodate to liberate iodine. The liberated iodine selectively oxidizes leucocrystal violet to crystal violet dye. The formed dye shows maximum absorbance at 590 nm. The color system obeys Beer's law in the concentration range from 0.4 - 3.6 microg antimony per 25 ml of final solution. The molar absorptivity and Sandell's sensitivity were found to be 7.32 x 10(5) l mol(-1) cm(-1) and 0.0016 microg cm(-2), respectively. All variables were studied in order to optimize the reaction. The proposed method is satisfactorily applicable for the analysis of antimony in various environmental and biological samples. The method is simple, highly sensitive, accurate and reliable.
