PhosphoSens-Red Kinase Assays 

Many kinase assays can be performed under general kinase reaction conditions. Our general kinase reactions include the following components.

• 54 mM HEPES, pH 7.5
• 1 mM ATP or ATP K_m
• 1.2 mM DTT
• 0.012% Brij-35
• 1% glycerol
• 0.22 mg/ml BSA
• 0.55 mM EGTA
• 10 mM MgCl₂
• 10-15 μM Sensor Peptide
• 0.05-5 nM Kinase (adjusted as needed)
• Additional co-factors or additives (as required)*

*Please reference the target-specific conditions table below to determine whether your target requires any additives or co-factors for optimal performance.

Some kinases require additives or co-factors for optimal performance. Please reference the target-specific conditions table below to see what, if any, additional reagents are recommended.

Prior to setting up the individual reactions, prepare the following solutions:

• PhosphoSens Sensor Peptide Substrate (1 mM):

Bulk PhosphoSens sensor peptides are supplied as 1 mg lyophilized powder. Dissolve the sensor peptide substrate as indicated on the vial or in the Technical Notes section of the Certificate of Analysis to create the 1 mM stock. If the sensor peptide requires the addition of dimethyl sulphoxide (DMSO), add the DMSO first to ensure solubility followed by the aqueous component, gently vortexing the solution between additions to ensure complete dissolution.  

Sensor peptides that are provided in the PhosphoSens Evaluation Kits, are already solubilized as a 1 mM solution in the solvents defined in the Technical Notes section of the Certificate of Analysis.

• PhosphoSens Sensor Peptide Substrate Solution (100 µM):

Sensor peptides that are provided in the PhosphoSens Kits, are already solubilized as a 1 mM solution in the solvents defined in the Technical Notes section of the Certificate of Analysis.

Prepare 0.1 mM Substrate solution by thawing the 1 mM peptide substrate stock solution, mixing well by vortexing gently, removing an appropriate amount and diluting 10-fold into ultrapure deionized water.

• Adenosine 5′-triphosphate disodium salt hydrate (ATP) Solution (10 mM):

ATP is supplied as a 100mM stock solution. Prepare 10 mM ATP solution by adding 50 μL of 100 mM ATP to 450 μL ultrapure deionized water.

NOTE: These instructions are for running the assay at 1 mM ATP. If running the assay at ATP K_m, please prepare 500 µL of a stock of ATP at 10X the ATP K_m.

• DL-Dithiothreitol (DTT) Solutions (10 mM):

DTT is supplied as 1M stock solution. Prepare 10 mM DTT solution by adding 5 μL of the 1 M DTT to 495 μL ultrapure deionized water.

CAUTION: Diluted DTT is readily oxidized so use fresh dilutions. 

• Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) Solution (5.5 mM):

EGTA is supplied as 550mM stock solution. Prepare 5.5 mM EGTA solution by adding 5 μL of 550mM EGTA to 495 μL ultrapure deionized water.

• Enzyme Reaction Buffer (ERB, 10X):

ERB is provided as a 10X stock solution. ERB is added directly to the ‘Master Mix’.

• Enzyme Dilution Buffer (EDB, 5X):

EDB is provided as a 5X stock solution. EDB is added to Blank or Background wells as a "No Enzyme" control and is used to dilute enzymes.

• Europium (30 mM):

Europium is provided as a 30 mM stock solution. This can be added directly to each well to a final concentration of 5 mM.

After preparing reagents as described above, prepare an appropriate amount of 'Master Mix' for your experiments.

The individual components and respective volumes to prepare 0.875mL of ‘Master Mix’ for a general kinase reaction at 1 mM ATP is outlined below:

Reaction Set-Up outlined below is written for a 20 μL total volume/well in 384-well plate and 10 μM sensor peptide:

Step 1. Add 2 μL 100 μM Sensor Peptide

Step 2. Add 14 μL ‘Master Mix’ containing reaction buffer, ATP, DTT, and EGTA (and additional co-factors or additives, if needed)

Step 3. Pause for 5-minute incubation at 30°C

Step 4. Add 4 μL 5X Enzyme dilution buffer or 5X Kinase in EDB

Step 5. Add plate to reader and monitor kinase activity by collecting fluorescence intensity (RFU) readings (lExMax 360 nm/lEmMax ~492 nm [485-498 nm]) every 0.5-2.0 minutes at 30°C until the progress curve of the no-inhibitor control reaches the top the linear range.

Step 6. Remove plate from reader and remove plate seal.

Step 7. If your target is a serine/threonine kinases, process to step 8.

If your target is a tyrosine kinase, add 5 µL of 0.5 M NaOH to each well and incubate for 5 minutes at room temperature to adjust the pH.  

Step 8. Add 4 µL of the 30 mM Europium solution to each well, for a final Europium concentration of 5 mM, and incubate for 5 minutes at room temperature.  

Step 9. Return the plate to the reader and take an endpoint time-resolved fluorescence (RFU) reading (lExMax 360 nm/lEmMax ~620 nm) with the plate unsealed.