Protein transformations that change lives

Services | ATP Competition Assay

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).

At the International Centre for Kinase Profiling (ICKP) we are happy to help you with all steps of your assay to provide you with the key information that can help move your studies forward.

When performing an enzymatic assay involving a kinase, one key factor may be determining the type of compound you are dealing with. Type I kinase inhibitors are ATP competitive – thus they target the ATP binding site of the kinase in its active conformation. In contrast, Type II kinase inhibitors are NOT ATP competitive. We can assist customers in determining whether their compound of interest is ATP competitive or not.

Workflow

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 K_m of an enzyme of interest (where feasible). Please contact us to discuss the ATP concentration range for this competitive assay (based on the known K_m 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

Data Analysis:

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

Submission Guidelines

Download: Compound Submission Form

All customers: Please provide a completed Compound Submission Form.

Academic customers are required to read the Terms & Conditions and indicate their agreement on the Compound Submission Form.
For-Profit customers are required to complete and return the Overarching Service Agreement before work can commence.

Amount:

Dry: Must be soluble in Dimethyl Sulfoxide (DMSO)
Wet:
A minimum of 0.02 ml of each compound in 100% DMSO
Concentration: 51 times the concentration at which the screen is to be carried out

Timeline

Rapid turnaround times are possible with these screens (1 week or less)
Please contact us to discuss your needs

Shipping to the UK

In order to minimize customs delays we encourage all customers to provide a sufficient amount of information on each package including:

Compound Name(s)
Derivation Source (synthetic, natural)
Intended Use (e.g. Research)

Shipping Address:

International Centre for Kinase Profiling Division of Signal Transduction Therapy
School of Life Sciences
University of Dundee
Dow Street
Dundee DD1 5EH
United Kingdom
Phone: +44-(0)1382-388-056

Services | ATP Competition Assay

Workflow

Submission Guidelines

Timeline

Shipping to the UK

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