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Setting up a kinase profiler with IMAP

termediate for subsequent steps.

c. Add 83 L of the intermediate 1:10 dilution of BR to the Binding Buffer to make a 1:1200 final dilution of the BR.

Step 4. Prepare 10 mL 40/60/1:2000 Binding Solution.

a. Mix 4 mL of 1X Buffer A with 6 mL 1X Buffer B to make Binding Buffer.

b. Add 50 L of the intermediate 1:10 dilution of BR prepared in Step B to the Binding Buffer to make a 1/2000 final dilution of the BR.

Step 5. Prepare 10 mL 10/90/1:1500 Binding Solution.

a. Mix 1 mL of 1X Buffer A with 9 mL 1X Buffer B to make Binding Buffer.

b. Add 67 L of the intermediate 1:10 dilution of BR prepared in Step B to the Binding Buffer to make a 1:1500 final dilution of the BR.

Step 6. Mix each Binding Solution thoroughly and add 60 L of the appropriate Binding Solution to the appropriate wells, as indicated in Table B.

Plate incubation
Incubate the plate in the dark overnight (16-24 hours) prior to reading the FP.

Note: Those wells to which Binding Solutions containing =<25% Buffer B were added may be read after a 1- hour incubation. Those assays with Binding Solution containing >25% Buffer B will require a longer incubation time to achieve a stable signal. To simplify the protocol, you can incubate the entire Profiler assay overnight and then read the FP just once.

Read in FP mode
Settings for the MDC Analyst AD, HT or GT:

  • Continuous lamp

  • FAM
    Excitation filter: 485nm-20fwhm
    Emission filter: 530nm-25fwhm

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