We can assist customers in determining whether their compound of interest is ATP competitive by analysing the effectiveness at 5 different concentrations of ATP. This analysis factors in 2 ATP concentrations on either side of the Km of an enzyme of interest (where feasible). Please contact us to discuss the ATP concentration range for this competitive assay (based on the known Km of your enzyme of interest).
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)
- Compounds are screened in duplicate
2. 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
3. Assay components are harvested onto P81 filter plate
4. Filter plates are air-dried
5. Scintillation fluid is added to plates
6. Counts are read on a Topcount NXT
1. Bar codes assigned to each file ensure that data corresponding to the correct compound is being analysed
2. A mean percentage activity is calculated for the duplicate assays for each customer.
- This provides a standard deviation for all the duplicates at each ATP concentration
Each customer receives mean percentage activity and standard deviation for
each compound tested against all enzymes