{"id":8837,"date":"2024-07-01T15:48:47","date_gmt":"2024-07-01T13:48:47","guid":{"rendered":"https:\/\/www.igmm.cnrs.fr\/8783-2\/"},"modified":"2024-07-19T17:32:15","modified_gmt":"2024-07-19T15:32:15","slug":"8783-2","status":"publish","type":"page","link":"https:\/\/www.igmm.cnrs.fr\/en\/8783-2\/","title":{"rendered":"Available proteins *"},"content":{"rendered":"
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Procedures and quality controls :<\/h3>\n
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TEV protease\u00a0:<\/u><\/strong><\/p>\n

The protease was expressed using the 6His-pTEV plasmid (backbone pET28 - map available on request), in the E.coli<\/em> Rosetta pLysS strain. It was then affinity purified on a Ni-NTA column, followed by anion exchange after desalting. The pure fraction was centrifuged to remove aggregates and stored in 50% glycerol.<\/p>\n

Its activity was then tested using Cas9-GFP, which features a TEV site between the 6H-MBP tag and Cas9-GFP.<\/p>\n

Cleavage efficiency depends on the accessibility of the enzyme binding site and can therefore vary a lot from one substrate to another. We recommend adapting the amount of TEV to the substrate, using a range of decreasing amounts of TEV starting at 1 \u00b5g TEV for 1 \u00b5g substrate.<\/p>\n

Purity analysis (Coomassie \u2013 10% acrylamide)\u00a0 :<\/strong><\/p>\n

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Cas9-GFP<\/u><\/strong><\/p>\n

The protein was expressed from the 6His-MBP-Cas9-GFP construct (addgene pMJ922) in the E.coli<\/em> Rosetta pLysS strain. It was purified via the following steps:<\/p>\n