Application
Functional Studies
Recombinant MYC (Abcam ab169901) at 1000 nM was incubated with 50 nM recombinant CDK9 together with cyclin T1 required for kinase activity (Thermo Fisher PV4131) and 100 μM ATP for 30 min at 30°C. The reaction was stopped by adding SDS sample buffer and boiling for 5 min at 95°C. After separation by SDS-PAGE, the gel was stained for 30 min with SimplyBlue Coomassie and destained overnight in water. Protein bands corresponding to MYC were excised and the proteins were reduced, alkylated, and in-gel digested with trypsin overnight at 37˚C. Peptides were extracted and dried by vacuum centrifugation. Samples were analyzed by LC/MS/MS using an Easy nLC 1000 coupled to a QExactive HF mass spectrometer.
Using a kinase assay followed by mass spectrometry, we identified putative CDK9 phosphorylation sites on MYC.
Using a kinase assay followed by mass spectrometry, we identified putative CDK9 phosphorylation sites on MYC.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.
Devon Blake
Verified customer
投稿 Aug 10 2020