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Setting up a kinase profiler with IMAP

Dichroic mirror: 505nm

    Excitation filter: 530nm-25fwhm
    Emission filter: 590nm-20fwhm
    Dichroic mirror: 561nm

  • Z-height: 3mm

  • Attenuator: out

  • SmartRead or Comparator for AD or HT

  • Sensitivity: 0 for AD or HT

  • Integration time: 100,000 sec for AD or HT, 20,000 sec for GT

    Settings for the MDC SpectraMax M5:

  • Endpoint Mode

  • Fluorescein
    Excitation: 485 nm
    Emission: 530 nm
    Auto cut-off: 515 nm

    Excitation: 530 nm
    Emission: 590 nm
    Auto cut-off: 561

  • Readings per well: 30

  • PMT sensitivity: medium

  • Autocalibrate: On

  • Automix: Off

  • Settling Time: Off

    Analyze your results
    Step 1. Calculate the average background (= Buffer Only wells) for both S and P fluorescent intensity data.

    Step 2. Subtract the background value from both S and P raw data.

    Step 3. Calculate FP and plot FP against compound concentration.


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