Services | IC50 Determination

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. 

The effectiveness of your compound is a key determinant factor of potency.  After screening a panel of kinases (hyperlink to Premier, Express, Custom, Lipid), the next logical step may be to determine your compound’s IC50 -- the half maximal inhibitory concentration of your compound.  This can prove to be critical information for a researcher’s studies.  At the ICKP, we can easily work with any number of compounds to assist you with determining the IC50 values. 

Please contact us to discuss further details, analysis requirements and pricing.

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 0.05 ml of each compound in 100% DMSO
  • Concentration: 51 times the top concentration of the IC50 curve

From this, we will proceed with half-log serial dilutions

Number of Compounds:

  • If there are more than 30 compounds please contact the ICKP for additional details regarding submission

 

Workflow

Protein Kinases

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.

Lipid Kinases

The method utilized for lipid kinase screening is the Promega ADP Glo High Throughput Assay kit.   The principle of this assay is that  it is a measurement of the ADP generated as a function of the enzyme reaction.

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. Protein Kinases

  • 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 designated incubation time (for each enzyme @ RT)
  • Orthophosphoric acid - Assay is halted with this addition
  • Assay components are harvested onto P81 filter plate
  • Filter plates are air-dried
  • Scintillation fluid is added to plates
  • Counts are read on a Topcount NXT

3. Lipid Kinases

  • Promega ADP Glo HTS

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

Timeline

  • IC50 determinations can be determined as a follow-up to initial kinase panel screens
  • These studies can be completed within a few working days from receipt of request or receipt of concentrated stock compound

Pricing

  • We work with BOTH academia and industry
  • We offer competitive rates.
  • There will be discounts for larger projects (>40 compounds within the same screen)
  • Please contact us to discuss your requirements

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
College of Life Sciences
University of Dundee
Dow Street
Dundee DD1 5EH
United Kingdom
Phone: +44-(0)1382-388-056

Do you need any help? Please get in touch and we’ll be happy to lend a hand.