A UPF1–U15 RNA complex was formed on a preparative scale by mixing 100 µg UPF1 protein with a 1.2-fold molar excess of fluorescein-labeled U15 RNA, followed by size-exclusion chromatography on a 2.4 mL Superdex 200 column. The peak fraction containing both UPF1 and U15 RNA was used for the native gel analysis. An amount of 2 µg of individual proteins and 4 µg of complexes were used in each case. Proteins were mixed with a 1.2-fold molar excess of fluorescein-labeled U15 RNA and incubated at room temperature for 30 min. Thereafter, samples were reconstituted in native gel sample buffer and directly analyzed on a 4%–20% Tris-Glycine native gel (Thermo Fisher Scientific). Proteins were visualized by staining with Coomassie brilliant blue and the U15 RNA was detected by fluorescence scanning using a Typhoon scanner (GE Healthcare).
Tris glycine native gel
Manufactured by Thermo Fisher Scientific
The 4%–20% Tris-Glycine native gel is a laboratory equipment used for the separation and analysis of proteins in their native, non-denatured state. It is a polyacrylamide gel electrophoresis (PAGE) system that employs a Tris-glycine buffer system to maintain the proteins in their native conformation during the separation process.
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2 protocols using tris glycine native gel
1
Purification and Characterization of UPF1-U15 RNA Complex
2
Characterization of UPF1-U15 RNA Complex
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A UPF1-U15 RNA complex was formed on a preparative scale by mixing 100 µg UPF1 protein with a 1.2-fold molar excess of fluorescein-labelled U15 RNA, followed by size-exclusion chromatography on a 2.4 mL Superdex 200 column. The peak fraction containing both UPF1 and U15 RNA was used for the native gel analysis. 2 µg of individual proteins and 4 µg of complexes were used in each case. Proteins were mixed with a 1.2-fold molar excess of fluorescein-labelled U15 RNA and incubated at room temperature for 30 minutes. Thereafter, samples were reconstituted in native gel sample buffer and directly analysed on a 4-20% Tris-Glycine native gel (ThermoFisher Scientific). Proteins were visualised by staining with Coomassie Brilliant Blue and the U15 RNA was detected by fluorescence scanning using a Typhoon scanner (GE Healthcare).
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