PRODUCT

PhosphoSens-Kinetic Kinase Assays

Reaction conditions and experimental protocols for PhosphoSens-Kinetic Kinase Activity Assays.

Reaction Conditions and Setup

Kinase assays that provide a direct measure of catalytic function are paramount for pre-clinical drug discovery success. Although limitations exist in establishing an enzyme assay for a new kinases, AssayQuant is striving to reduce this limitation to enable the quantum improvement  needed to accelerate creation of more effective therapeutics. 

With this goal in mind, access information below to ensure you are performing your assay with under optimized conditions.

For target specific assay validation data, please inquire at support@assayquant.com.

PhosphoSensExtended_Continuousonly

 

General Kinase Reaction Conditions

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 Km
  • 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.

Target-Specific Reaction Conditions

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.

Target(s)
HGNC (Common) Name
Component Description Final Concentration of Additive in Assay
CAMK1 (CAMK1α) Calcium Calmodulin Solution, 10X
50 ng/µl Calmodulin, 4 mM CaCl2, 1 M Tris-HCL, pH 7.0, 1 M Tris-HCl, pH 7.5 in nuclease free water
5 ng/µl Calmodulin
0.4 mM CaCl2
5mM TRIS-HCl, pH7
5mM TRIS-HCl, pH 7.5
(-) 0.55 mM EGTA 
PNCK (CAMK1β)
CAMK1D (CAMK1δ)
CAMK1G (CAMK1γ)
CAMK2A (CAMK2α)
CAMK2B (CAMK2β)
CAMK2D (CAMK2δ)
CAMK2G (CAMK2γ)
CAMK4
CAMKK1
DAPK1
DAPK2 (DRP‐1)
DAPK3 (ZIPK)
DCLK1 (DCAMKL1)
DCLK2 (DCAMKL2)
EEF2K (eEF2K)
MYLK (MLCK)
MYLK2
PHKG1
PHKG2
EIF2AK4 (GCN2)
10µg/mL t-RNA
10µg/mL t-RNA
1µg/mL t-RNA
PDK1 (PDHK1)
KCI Solution, 100 mM
100mM  KCl in nuclease free water
10 mM KCl
PDK2 (PDHK2)
PDK3 (PDHK3)
PDK4 (PDHK4)
PRKAA1‐B1‐G1 (AMPKα1β1γ1)
10 mM AMP
10 mM AMP
1 mM
PRKAA1-B1-G2 (AMPKα1β1γ2)
PRKAA1-B1-G3 (AMPKα1β1γ3)
PRKAA1‐B2‐G1 (AMPKα1β2γ1)
PRKAA1-B2-G2 (AMPKα1β2γ2)
PRKAA1-B2-G3 (AMPKα1β2γ3)
PRKAA2‐B1‐G1 (AMPKα2β1γ1)
PRKAA2-B1-G2 (AMPKα2β1γ2)
PRKAA2-B1-G3 (AMPKα2β1γ3)
PRKAA2‐B2‐G1 (AMPKα2β2γ1)
PRKAA2-B2-G2 (AMPKα2β2γ2)
PRKAA2-B2-G3 (AMPKα2β2γ3)
PRKCA (PKCα)
PS/DAG Solution, 10X
1.4 mM L-α-phosphatidylserine , 0.038 mM 1-2-dioleoyl-sn-glycerol, 1 M HEPES, pH 7.5 in nuclease free water
140 µM Phosphatidylserine
3.8 µM Diacylglycerol
(-) 0.55 mM EGTA
PRKCB1 (PKCβ1)
PRKCB2 (PKCβ2)
PRKCD (PKCδ)
PRKCE (PKCe)
PRKCG (PKCγ)
PRKCH (PKCη)
PRKCI (PKCι)
PRKCQ (PKCᶿ)
PRKCZ (PKCζ)
PRKDC (DNA‐PK)
500µg/mL DNA
500µg/mL DNA
1µg/mL t-RNA
PRKG1 (PKG1, PGK)
cGMP Solution, 10 mM
10 mM cGMP in nuclease free water
1 mM cGMP
PRKG2 (PKG2, PGK2, CGK2)
RIPK1
MgCl₂ Solution, 100mM 
100 mM MgCl₂ in nuclease free water
10 mM MgCl₂
RIPK2
RIPK3
RIPK5
BCKDK
KCI Solution, 500 mM
500mM  KCl in nuclease free water
50 mM KCl
MAP3K6
N/A
N/A
(-) 1.2 mM DTT

Preparing Reagents

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 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 Km, please prepare 500 µL of a stock of ATP at 10X the ATP Km.

  • 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.

Preparing ‘Master Mix’

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

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

COMPONENT

VOLUME

Enzyme Reaction Buffer (10X)

125 μL

ATP Solution (10 mM)   

125 μL

DTT Solution (10 mM)   

125 μL

EGTA Solution (5.5 mM)

125 μL

Water

375 μL

 

PhosphoSens-Kinetic Kinase Reaction Set-Up

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.

Experiment Specific Protocols

PROTOCOL

PhosphoSens-Kinetic Kinase Inhibitor IC50 Determination 

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