PhosphoSens-Kinetic Phosphatase Assays

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

• 54 mM HEPES, pH 7.5
• 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 Phosphatase (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 phosphatases 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 1mg 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 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 Evaluation 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.

• 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.

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 phosphatase reaction 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, 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 Phosphatase in EDB

Step 5. Add plate to reader and monitor phosphatase 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.