Kinase SubstrateFinder Assay Service

Identifying a suitable substrate for a given protein kinase is often a difficult task. Sometimes the physiological substrate of a protein kinase is unknown or does not work under in vitro conditions in a screening assay. Also, some protein kinases will not accept short peptides as substrates but require proteins. 

Reaction Biology offers a collection of 58 generic serine/threonine- and tyrosine protein kinases substrates that are comprised of proteins, artificial fusion proteins, generic polypeptides, and peptides.

Based on our experience our collection of substrates includes at least one that can be identified to be suitable for in vitro kinase assay development.

We will verify identified substrates in customized assay setups including the determination of enzymatic parameters V_MAX and ATP K_m and/or substrate K_m.
 

Example of MELK assay development with the Generic Kinase SubstrateFinder.

MELK kinase activity was measured with a variety of generic substrates. Controls are no-enzyme controls of each substrate and autophosphorylation of the MELK kinase without substrate. Substrate 14 is the most suitable substrate for the establishment of a MELK assay. 
 

Setups: Singlicate screening with three substrate concentrations. 

Assay format: ADP-Glo assay (Promega)

ATP concentrations: 10 µM ATP

Controls: Background and autophosphorylation control. Two control peptides for positive controls provided by the customer. 

Turnaround time: 10 business days for standard projects. Expedited scheduling and data delivery can be arranged prior to the commencement of the studies. 

Report: The report includes a table of all substrates ranked according to kinase activity, a graphical presentation of the ranking, the identity of the 3 “top scorers”, and a comparison to two control substrates (if provided by the customer).

Further testing: The Kinase SubstrateFinder can be used as a stand-alone service or be part of an assay development package. We can verify identified substrates in customized assay setups including determination of enzymatic parameters V_MAX and ATP K_m and/or substrate K_m.

Sample requirements: In brief, for a standard project, we require 15 µg of the purified kinase. Kinase stock solution concentration should be at least 100 µg/ml.

Your kinase of interest is screened against a library of biotinylated, physiological relevant peptides selected from the SwissProt and Phosphobase databases. 
Information you can gain from the Physiological SubstrateFinder service include:

• Determination of protein kinase substrate consensus sequences
• Identification of potential physiological substrates/downstream targets
• Investigation of signal transduction pathways
• Identification of protein kinase peptide substrates for screening purposes
   

Example of CDK1/CycB1 testing with 720 physiological substrates. 

Upper left: ranking of all tested peptides from two runs. Upper right: Activity of two control substrates, autophosphorylation (auto) and background (BG) as well as the kinase activity with the 5 best substrates. Table: Listing of the top 5 substrates identified from the substrate library screen. 

Setups: Single point screening with one peptide concentration.  

Assay format: Radiometric ³³PanQinase™ assay

ATP concentrations: 1µM ATP

Controls: Background and autophosphorylation control. Two control peptides for positive controls provided by customer. 

Turnaround time: 10 business days for standard projects. Expedited scheduling and data delivery can be arranged prior to commencement of the studies. 

Report: The raw data and graphical ranking of all peptides is included in the report.  Graphical presentation of controls and rank 1 to 5 peptides will be provided beside detailed material and methods. 

Screening location: The assay is performed in Freiburg, Germany. 

Sample requirements: In brief, for a standard project we require 23 µg of purified kinase. Kinase stock solution concentration should be at least 100 µg/ml.