Protein Kinase A Activity Assay Measured in Biochemical System Using Plate Reader - 2052-04_Inhibitor_Dose_DryPowder_Activity
Assay Overview: Inhibition of protein kinase A (PKA) activity is measured using recombinant His tagged PKA catalytic fragment and a peptide substrate in an end-point readout assay. Purified PKA is mixed together with ATP, Mg ions, fluorescently labeled peptide substrate and various concentrations of compounds of interest. The enzymatic reaction is allowed to proceed and then stopped by the addition of the metal chelator EDTA. Conversion of substrate to product is measured using a Caliper Life Sciences capillary electrophoresis lab on chip device. ..more
BioActive Compounds: 3
Keywords: PKA, protein kinase A, cAMP dependent Kinase
Assay Overview: Inhibition of protein kinase A (PKA) activity is measured using recombinant His tagged PKA catalytic fragment and a peptide substrate in an end-point readout assay. Purified PKA is mixed together with ATP, Mg ions, fluorescently labeled peptide substrate and various concentrations of compounds of interest. The enzymatic reaction is allowed to proceed and then stopped by the addition of the metal chelator EDTA. Conversion of substrate to product is measured using a Caliper Life Sciences capillary electrophoresis lab on chip device.
Expected Outcome: Non-selective STK33 inhibitors will also inhibit PKA while selective ones will not. IC50s for compounds of interest are determined based on %conversion vs compound concentration and are used to decide which compounds are selective for STK33 or inhibit both kinases.
Assay Buffer (AB)
10 mM MOPS pH 7
300 uM EDTA
0.5 % (V/V) Glycerol
0.001% (V/V) Brij-35
In a 384 well polypropylene plate
+ 20 uL / well 15 uM ATP, 15 mM MgAc2 in AB
+ 25 nL compound dilutions in DMSO using a pin tool
+ 5 uL 5 nM PKA, 15 uM Peptide 21 in AB
incubate 1 hr RT
1.5 nM PKA, 3 uM Peptide 21 (Fluorescent Peptide Substrate), 12 uM ATP (final conditions)
stop enzyme reaction
+ 10 uL 75 mM EDTA in AB
Determine % conversion of peptide to phospho-peptide using Caliper capillary electrophoresis instrument at the conditions specified by the manufacturer for Peptide 21.
Reported Km ap (ATP) 6 uM
Measure Km ap (Pep21) 4.6 uM
A high ATP concentration assay may be run identically to above save with final concentrations of 0.3 nM PKA, 120 uM ATP as a preliminary test of whether or not an inhibitor is ATP competitive.
PRESENCE OF CONTROLS: were included on every plate.
EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
The compounds were assayed in multiple independent instances using an identical protocol; each instance is called a 'test'. For each test, the following analysis was applied:
ACTIVE CONCENTRATION LIMIT:
For each sample, the highest valid tested concentration (Max_Concentration) was determined and the active concentration limit (AC_limit) was set to equal (10)(Max_Concentration).
No normalization was applied to the raw data signals.
PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.7.0.3) was applied.
MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): AC50
AC values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).
AC values were calculated up to the active concentration limit described for each sample.
pAC was set to equal -1*log10(AC)
Activity_Outcome = 1 (inactive) when:
a) compound shows activity but in a direction opposite to the expected outcome
in these cases, values describing curve fitting parameters (Sinf, S0, Hill Slope, log_AC50, log_AC50_SE) are set to null
b) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
c) AC > AC_limit
Activity_Outcome = 2 (active) when:
AC <= AC_limit
Activity_Outcome = 3 (inconclusive) when:
a) Curve fitting strategy resulted in a constant fit with activity >= -70% but <= -30%, or
b) The fit was deemed not valid due to poor fit quality.
If PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive),
then PUBCHEM_ACTIVITY_SCORE = 0
If PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
then PUBCHEM_ACTIVITY_SCORE = (10)(pAC)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
When the active concentration (AC) is calculated to be greater than the highest valid tested concentration (Max_Concentration), the PUBCHEM_ACTIVITY_SCORE is calculated using Max_Concentration as the basis.
When the active concentration (AC) is calculated to be less than the lowest tested concentration, the PUBCHEM_ACTIVITY_SCORE is calculated using the lowest tested concentration as the basis.
Once the data for each test was processed, the test number was appended to all column headers in that test's data set. The individual test results were then aggregated as follows:
1. The final PUBCHEM_ACTIVITY_OUTCOME was set to:
a. '2' (active) when all test outcomes were '2' (active), or
b. '1' (inactive) when all test outcomes were '1' (inactive), or
c. '3' (inconclusive) when the test outcomes were mixed.
2. The final ACTIVE_CONCENTRATION (AC) was set as follows:
a. If the final PUBCHEM_ACTIVITY_OUTCOME = 2, AC was set as the mean of the constituent test active concentrations;
b. If the final PUBCHEM_ACTIVITY_OUTCOME = 1 or 3, AC was left empty.
3. The final PUBCHEM_ACTIVITY_SCORE was calculated based on the aggregated ACTIVE_CONCENTRATION, using the same logic described above for individual test scores.
The individual dose data point columns ('Activity_at_xxuM') reported here represent the median of valid (unmasked) replicate observations at each concentration. These values are the inputs to a curve fitting algorithm.
All other data columns represent values which are derived during the curve fitting algorithm; this may sometimes include automatic further masking of some replicate data points.
Occasionally this results in perceived inconsistencies: for example, between the derived 'Maximal_Activity' and the apparent most active data point.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)