User:Christine Doan/Notebook/Identifying Kinase Substrates of PfCK2/2014/03/18

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new CK2

  • Thaw 5ml pellet under running water
  • Add 5mg lysozyme directly to resuspended pellet
  • Incubate on ice for 15mins
  • Take 100ul, dilute to 1ml with MK buffer, take 250ul of that dilution, dilute to 1ml with MK buffer, measure OD
  • Sonicate 6x 15sec, 1min on ice in between
  • Take 100ul, repeat previous dilution step
    • Found that there was little to no lysis. Sonicator was set to 60% amplitude due to students from other labs using different settings
    • DC said to use the larger sonicator in the protein lab. Optimum regiment was found to be 30% amplitude, 6x 20sec, 30sec on ice in between
    • Desired "before" OD = 1, desired "after" OD = 0.2 (80% lysis)
  • Ultracentrifuge
    • Takes a long time to cool and pressurize - turn on ahead of time and keep rotor in cold room
    • Diluted ~4ml of resuspended pellet with 4ml MK buffer to fill tube ~75%
    • Make sure tubes are as closely balanced as possible!
    • Turn off vacuum to open door, insert samples, set rotor on peg, no grooves or markings to line up
    • Will spin up to 3000 before kicking up to desired speed
    • Spin 35K for 30mins
  • Transfer ~8ml sup to conical, store -80C
  • Resuspend pel in ~8ml MK buffer, took forever, store at 4C to dissolve

substrates

  • Still waiting on pET-30 Ek/LIC kit

In vitro kinase assay: Kinase-GLO Luminescence is correlated to amount of ATP and inversely correlated to kinase activity Can be used for kinases that phosphorylate on multiple sites Radiolabeled assay