This protocol was used in the above Nature Immunology paper.
Method Article
Kinase assays
https://doi.org/10.1038/nprot.2007.67
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This protocol was used in the above Nature Immunology paper.
2.Lyse cells in TNE (50 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 mM PMSF, and 1-5 mM Na3VO4. Clear lysates by centrifugation at 12,000 X g for 10 minutes at 4oC.
Set up immunopreciptations of Dlgh1 using 40 μl of protein G (50% slurry so 20μl bead volume) (GE Healthcare Bio-Sciences 17-0618-02) and 10 μl (2.5μg) of Dlgh1 (BD Transduction Lab 610875) or p38 (2.0μg) (Santa Cruz, C-20, sc-535) antibody. Tumble at 4uC for 1-2 hours. Save some of this supernatant to check efficiency of IP.
Wash once with 500μl of kinase buffer (25mM Tris pH 7.5, 5mM B-glycerophosphate, 2mM DTT, 0.1 mM Na3VO4 and 10mM MgCL2; Cell signaling 9802)
In a final volume of 50μl have 1μg of GST-ATF2 (Cell signaling, 9224S) and/or 50μM unlabeled ATP (Cell signaling, 9804) with immunopreciptates in kinase buffer. (20μl of beads, 1μl of ATP, 29 μl of kinase buffer) MAKE SURE TO DO ALL OF THIS ON ICE
Incubate at 30oC for 5-20 minutes.
Add 20μl 5X Loading buffer to stop reaction and boil for 5 minutes
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
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