Overview
This information is relevant for LANCE™ Ultra kinase assays that use a ULight™-labeled substrate (2-component assay). All concentrations listed represent the final concentration in the reaction; all assays in triplicate:
1. Substrate identification/selection (if applicable)
- Kinase reaction (10 µL)
- For Ser/Thr kinases, use 20 nM kinase
- For Tyr kinases, use 1 nM or less kinase (a low concentration will likely need to be used, due to possible generic anti-phosphotyrosine antibody depletion).
- 50 or 100 nM ULight-substrate (refer to the tech data sheet for the product to see what concentration to use)
- 100 µM ATP
- Control: no ATP
- Incubate 60 minutes
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Detection (brings volume to 20 µL)
- 10 mM EDTA
- 2 nM Europium antibody
Figure 1. Figure (from Ser/Thr KinaSelect™ manual). Selection of the optimal substrate for the Aurora A kinase. The Aurora A kinase (Carna Biosciences) at 20 nM was incubated with either ULight-CREBtide (Ser133), ULight-PLK (Ser137), ULight-MBP, ULight-Histone H3 (Thr3) or ULight p70 S6K (Thr389) peptide in the presence of 200 µM ATP. Kinase reactions were terminated after 1 hour by the addition of EDTA followed by the addition of 1X LANCE Detection buffer containing the corresponding Eu-labeled antibody at a final concentration of 2 nM. Signal was read after 1 hour. A) LANCE signal at 665 nm. B) S/B ratio: +ATP/-
2. Enzyme titration vs. time-course kinetics
- Choose 4-6 concentrations of kinase
- For Ser/Thr kinases, appropriate kinase concentrations will generally range from 10 pM to 20 nM (final concentration in 10 µL kinase reaction)
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For Tyr kinases, choose concentrations near and below the concentration listed in the Tyrosine kinase substrate selection guide. In general, your range will be below 1 nM (final concentration in 10 µL kinase reaction). See below for generic anti-phosphotyrosine antibody depletion.
- Run time course experiment at each concentration of kinase (0, 5 min, 15 min, 30 min, 60 min, and 90 min). This is the time course for the kinase reaction step. You will stop the kinase reaction by adding EDTA. For the 0 min time point, add kinase + substrate + EDTA, incubate 1 minute, then add ATP (only after the EDTA has been added).
- 100 µM ATP; 50 or 100 nM ULight-substrate (refer to the tech data sheet for the product to see what concentration to use), 2 nM Eu-anti-phospho-antibody.
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Control: 0 nM kinase
Figure on left (from Tech note): Aurora B enzyme was incubated at concentrations ranging from 10 to 250 pM with 50 nM ULight-Histone H3 peptide and 20 µM ATP. Kinase reactions were terminated after 0 to 120 minutes by the addition of EDTA. The anti-phospho-Histone H3 (Ser10) antibody was used. Figure on the right: mTOR enzyme was incubated at concentrations ranging from 1.25 to 20 nM with 50 nM ULight-p70 S6K peptide and 200 µM ATP. Kinase reactions were terminated after 0 to 120 minutes by the addition of EDTA.
3. ATP titration
- ATP concentrations: 300 µM, 100 µM, 30 µM, 10 µM, 3 µM, 1 µM, 300 nM, 100 nM, 30 nM, 10 nM, and 0 nM
- Choose a kinase concentration and time point from your previous data. You will want to pick a concentration and time point in the linear range of your graph that give good S:B while conserving kinase
- 50 or 100 nM ULight-substrate (refer to the tech data sheet for the product to see what concentration to use), 2 nM Europium antibody
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Determine Km for ATP
4. Inhibition curve
- ATP concentration at or below the Km determined above
- Same kinase concentration and time point used in your ATP titration experiment
- Inhibitor concentrations as relevant
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50 or 100 nM ULight-substrate (refer to the tech data sheet for the product to see what concentration to use), 2 nM Europium antibody
Figure from Tech note
5. Z' factor determination
In a 384-well OptiPlate™, set up one row with kinase + substrate + ATP as optimized above + inhibitor (or exclude ATP). Set up another row with kinase + substrate + ATP as optimized above + 2% DMSO or buffer.
For research use only. Not for use in diagnostic procedures.
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