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Characterising Maturation of GFP and mCherry of Genomically Integrated Fusions in Saccharomyces cerevisiae.

Bio-protocol (2018-02-13)
Sviatlana Shashkova, Adam Jm Wollman, Stefan Hohmann, Mark C Leake
RESUMEN

Single-molecule fluorescence microscopy enables unrivaled sub-cellular quantitation of genomically encoded fusions of native proteins with fluorescent protein reporters. Fluorescent proteins must undergo in vivo maturation after expression before they become photoactive. Maturation effects must be quantified during single-molecule analysis. Here we present a method to characterise maturation of GFP and mCherry genetic protein fusions in budding yeast Saccharomyces cerevisiae.

MATERIALES
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Sigma-Aldrich
Cicloheximida, from microbial, ≥94% (TLC)
Millipore
Yeast Nitrogen Base Without Amino Acids and Ammonium Sulfate, NutriSelect® Basic, powder, for fungi, poly bottle of 500 g, poly bottle of 1 kg