The principal method utilized is a radioactive filter binding assay using 33P ATP (Hastie, et al 2006. Nat Protoc. 2006;1(2):968-71; Bain, et al 2007. Biochem J. 2007 Dec 15;408(3):297-315). This method is sensitive, accurate and provides a direct measure of activity.
Assay step-by-step process:
1. Upon receipt of your small molecule, staff at the ICKP will dilute each to the appropriate concentration (if required)
2. This compound is added to a ‘mother plate’ consisting of customer samples, controls and blanks
- These serve as the source for ‘daughter plates’ which are stored at -20* until assay initiation
- Note: All compounds are screened in duplicate
3. Next there will be 3 additions to the assay:
- Enzyme-substrate mixture
- Incubation Time: 5 minutes at Room Temperature (RT)
- 33P ATP - Assay begins with this addition
- Incubation time varies based upon optimal designatedincubation time(for each enzyme @ RT
- Orthophosphoric acid - Assay is halted with this addition
4. Assay components are harvested onto P81 filter plate
5. Filter plates are air-dried
6. Scintillation fluid is added to plates
7. Counts are read on a Topcount NXT
Data Analysis:
1. Bar codes assigned to each file ensure that data corresponding to the correct compound is being analysed
2. After completion of each assay, ICKP staff ensure that the run has passed standard quality control measures by examining reference compounds on the QC plate
3. Upon determination that the run has met QC standards, a Z-Prime (Z’) value is calculated utilizing data from the controls/blanks on each individual plate
- This QC measure is in place to ensure that each individual plate in the run has passed QC
4. Finally, a mean percentage activity is calculated for for each customer.
- A standard deviation for all the duplicates is also calculated
Each customer receives mean percentage activity and standard deviation for
each compound tested against all enzymes
There are over 500 kinases that have been identified in the human kinome comprising 518 protein kinases, and approximately 20 lipid kinases (Manning, et al. 2002. Science 298(5600):1912-34).