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_93
Snippet: Mass measurements were performed on a Viscotek MALS-20 and VE3580 RI detector attached to an ÄKTA 10 Purifier with microflow components. Protein samples were run at approximately 0.75 mg/ml on a Superdex200 Increase 10/300 GL column, running at 1.0 ml/min in gel exclusion buffer (with 0.5 mM TCEP instead of DTT) with 100-µl injections. per well and incubation for 15 minutes at room temperature. Two microliters of protein from each well was spot.....
Document: Mass measurements were performed on a Viscotek MALS-20 and VE3580 RI detector attached to an ÄKTA 10 Purifier with microflow components. Protein samples were run at approximately 0.75 mg/ml on a Superdex200 Increase 10/300 GL column, running at 1.0 ml/min in gel exclusion buffer (with 0.5 mM TCEP instead of DTT) with 100-µl injections. per well and incubation for 15 minutes at room temperature. Two microliters of protein from each well was spotted directly onto a nitrocellulose membrane, followed by western blotting, as described above. Selected clones were seeded from the second 96-well plate into 6-well plates, grown and TRIM25 levels were validated by standard western blotting.
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