Author: Nila Roy Choudhury; Gregory Heikel; Maryia Trubitsyna; Peter Kubik; Jakub Stanislaw Nowak; Shaun Webb; Sander Granneman; Christos Spanos; Juri Rappsilber; Alfredo Castello; Gracjan Michlewski
Title: RNA-binding activity of TRIM25 is mediated by its PRY/SPRY domain and is required for ubiquitination Document date: 2017_10_9
ID: ifla4aix_68
Snippet: EMSAs were performed with an end-labeled pre-let-7a-1 probe (50×10 3 c.p.m. (counts per minute), ~0.1 pmol) and the indicated amounts of proteins produced in E. coli. Binding reactions were performed in 16 μ l reactions containing gel exclusion buffer (4.8 mM Tris pH 8, 144 mM NaCl, 0.96 mM DTT) supplemented with 3 mM MgCl 2 , 0.5 mM ATP, and 37.5 mM creatine phosphate. Reactions were incubated at 4°C for 1 h, followed by electrophoresis on a .....
Document: EMSAs were performed with an end-labeled pre-let-7a-1 probe (50×10 3 c.p.m. (counts per minute), ~0.1 pmol) and the indicated amounts of proteins produced in E. coli. Binding reactions were performed in 16 μ l reactions containing gel exclusion buffer (4.8 mM Tris pH 8, 144 mM NaCl, 0.96 mM DTT) supplemented with 3 mM MgCl 2 , 0.5 mM ATP, and 37.5 mM creatine phosphate. Reactions were incubated at 4°C for 1 h, followed by electrophoresis on a 6% (w/v) non-denaturing polyacrylamide gel. The signal was recorded with radiographic film or as exposed to a phosphoimaging screen and scanned on a FLA-5100 scanner (Fujifilm).
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