Products/Services Used | Details | Operation |
---|---|---|
Mutagenesis Services | GFP-IMP1KH1-4mut construct was obtained by site-directed mutagenesis of the GXXG loops in the four KH domains (17) from GK(E/K/G)G to GELG. YBX1 was cloned into pcDNA3.1 + N-eGFP (Genscript) inserting a 25 aminoacid flexible linker (5× GGGGS) (18) between the fluorescent tag and YBX1. IMP1 and YBX1 (including flexible linker) coding sequences were also cloned into pmCherry-C1 (Clontech) | Get A Quote |
Understanding the mRNA life cycle requires information about the dynamics and macromolecular composition and stoichiometry of mRNPs. Fluorescence correlation and cross-correlation spectroscopy (FCS and FCCS) are appealing technologies to study these macromolecular structures because they have single molecule sensitivity and readily provide information about their molecular composition and dynamics. Here, we demonstrate how FCS can be exploited to study cytoplasmic mRNPs with high accuracy and reproducibility in cell lysates. Cellular lysates not only recapitulate data from live cells but provide improved readings and allow investigation of single mRNP analysis under particular conditions or following enzymatic ... More