The high-throughput screening of 210,000 compounds was conducted in 384-well plates using Kinase-Glo ® (Promega Inc.). Seven hundred and forty-one plates were screened, 36 plates per day, in the UT Southwestern High-Throughput Screening Core (HTS). The substrate solution was comprised of 55.3 mM HEPES (pH 7.4), 55.3 mM MgCl2, 4.7 µM pWNK1, and 19.3 µM GST-OSR1. Fifteen microliters of it was added to each well by multiplexed dispensing (Multidrop384, Thermo Scientific). Then, 0.2 µL of pure DMSO (negative control, columns 2 and 23) or 0.5 mM compound (columns 3 to 22) or 0.4 mM control quinazoline inhibitor (Figure S1A
Kinase glo
Manufactured by Promega
Sourced in Germany, United States
Kinase-Glo is a bioluminescent assay that measures the amount of ATP remaining in a kinase reaction. It provides a simple, sensitive, and homogeneous method for the detection of kinase activity.
Automatically generated - may contain errors
Lab products found in correlation
Pnk buffer, by New England Biolabs (2 mentions)
Eif2α peptide substrate, by Santa Cruz Biotechnology (2 mentions)
Multidrop 384, by Thermo Fisher Scientific (1 mentions)
Envision multilabel plate reader, by PerkinElmer (1 mentions)
Synergy ht plate reader, by Agilent Technologies (1 mentions)
D luciferin, by Merck Group (1 mentions)
Recombinant firefly luciferase, by Merck Group (1 mentions)
Pherastar plate reader, by BMG Labtech (1 mentions)
Kinase glo kit, by Promega (1 mentions)
Fluoroskan ascent fl, by Thermo Fisher Scientific (1 mentions)
Glomax96 microplate reader, by Promega (1 mentions)
Phosphoglycogen synthase peptide 2, by Merck Group (1 mentions)
Adenosine triphosphate (atp), by Cell Signaling Technology (1 mentions)
Envision 2, by PerkinElmer (1 mentions)
Bovine serum albumin (bsa), by Avantor (1 mentions)
Luminometer model td20 20, by Turner Designs (1 mentions)
Td 20 20, by Turner Designs (1 mentions)
Fluoroskan ascent fl fluorescence plate reader, by Thermo Fisher Scientific (1 mentions)
Plartlsvaglpgkk, by Bachem (1 mentions)
Flx800, by Agilent Technologies (1 mentions)
21 protocols using kinase glo
1
High-Throughput Screening of Kinase Inhibitors
2
Purification and ATPase Assay of ClpX
3
Luminescence-Based Luciferase Inhibition Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Confirmed qHTS active compounds were tested in a biochemical assay to directly measure potential for luciferase inhibition using Kinase-Glo (Promega). Briefly, 3 μl of substrate buffer (50 mM Tris–HCl [pH 7.5], 10 mM MgCl2, 0.05% bovine serum albumin, 0.01% Tween-20, 13.33 μM d -luciferin (Sigma; catalog no.: L9504), and 13.33 μM ATP) were dispensed into 1536-well assay plates. Compounds were then transferred via Kalypsys pin tool equipped with 1536-pin array. Following addition of compound, 1 μl of recombinant firefly luciferase (Sigma; L9506 prepared in 50 mM Tris–HCl [pH 7.5], 10 mM MgCl2, 0.05% bovine serum albumin, and 0.01% Tween-20; 10 nM final) was added to initiate the reaction. After 10 min of room temperature incubation, end-point measurements of luminescence were acquired using a ViewLux plate reader equipped with clear filters.
4
Kinase Inhibition Assay for PfCDPK1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
IC50 determinations were performed
using Kinase Glo (Promega) to measure
ATP depletion resulting from the kinase reaction. Compounds were added
to 22 μL reaction mixtures in white 384-well plates containing
10 nM full length recombinant PfCDPK1 and 8 μM
MyoA-Tail domain Interacting Protein (MTIP) in assay buffer (Tris-HCl
buffer at pH 8.0 containing 0.1 mM EGTA, 0.2 mM CaCl2,
1 mM DTT, and 0.01% Triton X-100) and incubated for 30 min at rt prior
to initiating the reaction with 10 μM ATP (Km) and 20 mM MgCl2 at final concentrations. Reactions were allowed to proceed
for 120 min at ambient temperature and stopped by the addition of
22 μL of Kinase Glo Plus detection reagent. Luminescence proportional
to the remaining ATP at the end of the reaction was measured using
a Pherastar plate reader (BMG Labtech). Ten point dose–response
curves were obtained from half-log dilutions of the test compound
diluted in assay buffer at a constant final DMSO concentration of
1%.
using Kinase Glo (Promega) to measure
ATP depletion resulting from the kinase reaction. Compounds were added
to 22 μL reaction mixtures in white 384-well plates containing
10 nM full length recombinant PfCDPK1 and 8 μM
MyoA-Tail domain Interacting Protein (MTIP) in assay buffer (Tris-HCl
buffer at pH 8.0 containing 0.1 mM EGTA, 0.2 mM CaCl2,
1 mM DTT, and 0.01% Triton X-100) and incubated for 30 min at rt prior
to initiating the reaction with 10 μM ATP (Km) and 20 mM MgCl2 at final concentrations. Reactions were allowed to proceed
for 120 min at ambient temperature and stopped by the addition of
22 μL of Kinase Glo Plus detection reagent. Luminescence proportional
to the remaining ATP at the end of the reaction was measured using
a Pherastar plate reader (BMG Labtech). Ten point dose–response
curves were obtained from half-log dilutions of the test compound
diluted in assay buffer at a constant final DMSO concentration of
1%.
5
Quantitative NDPK Activity Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HUVECs or retinae were lysed in RIPA buffer and immediately subjected to the NDP kinase activity assay. The test utilizes the transphosphorylase activity of NDPK to generate ATP from GTP and ADP. The newly-formed ATP is directly used by firefly luciferase to convert D-luciferin to the light-emitting oxyluciferin with the Kinase-GLo kit (V6711, Promega, Germany). Under the experimental conditions, the luminescent signal is proportional to the amount of ATP produced, and thus to the enzymatic activity of NDPK. A standard curve was generated by adding increasing concentrations of ATP (0.5–10 µM) in the absence of NDPKs. The NDP kinase activity in cell lysates was measured in a 384-well plate. Lysates were diluted in assay buffer consisting of 50 mM Tris-HCl, pH 7.5, 2 mM MgCl2, 1 mM dithiothreitol (DTT), and 0.01% bovine serum albumin (BSA). The readout was commenced by adding the luciferase substrate (V6711, Kinase-GLo, Promega, Germany). After 5 min of incubation, the luminescence was recorded at room temperature using a multiplate reader (EnVision, PerkinElmer, Germany).
6
MTH1 Inhibition Assay Protocol
7
Kinase Activity Assay for GSK3β
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The kinase activity assay was performed as previously described [17 (link)]. Briefly, 20 ng of recombinant human GSK3β (Biomol, Hamburg, Germany) were incubated with the substrate phospho glycogen synthase peptide 2 (pGS2, 25 μM) (Millipore, Billerica, USA), ATP (1 μM) (Cell Signaling, Frankfurt am Main, Germany) and different concentrations of PDA-66 and SB-216763 for 30 min at 30°C. After addition of Kinase-Glo (Promega, Mannheim, Germany) and 10 min of incubation at room temperature the luminescence signal was measured with a Glomax 96 microplate reader (Promega).
8
Kinetic Analysis of GST-CLK2 Variants
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Purified GST-CLK2cd, GST-CLK2cdK193A, and GST-CLK2cdK193A/D290A
at
14.5, 72.46, and 362.32 nM, respectively, were incubated with 2-fold
serial dilutions of a mixture of ATP (Sigma, catalog no. A7699) and
S6K peptide (KRRRLASLR) (SignalChem, catalog
no. S05-58) in a 1:5 ratio (ATP:S6K) from 20 to 0.15 μM in 10
μL of kinase assay buffer A [25 mM Tris-HCl (pH 7.5), 0.1 mg/mL
BSA, 10 mM MgCl2, 0.5 mM NaO3V, 5 mM β-glycerophosphate,
and 2 mM DTT] in white 384-well plates (Greiner bio-one, catalog no.
781904) for 1 h at 30 °C. Ten microliters of Kinase-Glo (Promega,
catalog no. V6713) was then added to the reaction mixture and incubated
for 5 min at room temperature before the luminescence was read in
the PerkinElmer Envision II instrument. The initial velocity (molar
per second, y axis) was defined as the change in
the concentration of ATP used for phosphorylated S6K, which was plotted
versus the concentration of substrate ATP before reaction (molar, x axis). The curves were then fitted using the nonlinear
regression method in R software, from which the Vmax and Km (Michaelis constant)
for each enzyme–substrate reaction were derived. The kcat (catalytic constant) was determined by dividing Vmax by the enzyme concentration. The catalytic
efficiency is defined as kcat/Km (inverse molar liter per second).
at
14.5, 72.46, and 362.32 nM, respectively, were incubated with 2-fold
serial dilutions of a mixture of ATP (Sigma, catalog no. A7699) and
S6K peptide (KRRRLASLR) (SignalChem, catalog
no. S05-58) in a 1:5 ratio (ATP:S6K) from 20 to 0.15 μM in 10
μL of kinase assay buffer A [25 mM Tris-HCl (pH 7.5), 0.1 mg/mL
BSA, 10 mM MgCl2, 0.5 mM NaO3V, 5 mM β-glycerophosphate,
and 2 mM DTT] in white 384-well plates (Greiner bio-one, catalog no.
781904) for 1 h at 30 °C. Ten microliters of Kinase-Glo (Promega,
catalog no. V6713) was then added to the reaction mixture and incubated
for 5 min at room temperature before the luminescence was read in
the PerkinElmer Envision II instrument. The initial velocity (molar
per second, y axis) was defined as the change in
the concentration of ATP used for phosphorylated S6K, which was plotted
versus the concentration of substrate ATP before reaction (molar, x axis). The curves were then fitted using the nonlinear
regression method in R software, from which the Vmax and Km (Michaelis constant)
for each enzyme–substrate reaction were derived. The kcat (catalytic constant) was determined by dividing Vmax by the enzyme concentration. The catalytic
efficiency is defined as kcat/Km (inverse molar liter per second).
9
Purification and Characterization of PGAM Enzymes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Full-length, histidine-tagged iPGAM from C. elegans and B. malayi and full-length, histidine-tagged dPGAM from Homo sapiens and Plasmodium falciparum were expressed and purified as described previously [7] (link), [8] (link), [23] (link). 3-PG, adenosine diphosphate (ADP), nicotinamide adenine dinucleotide (NADH), PyK (from rabbit muscle, product P7768), LDH (from rabbit muscle, product L2500) and a PyK/LDH mixture (from rabbit muscle; product P0294) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Enolase (from yeast; product #15515) was procured from Affymetrix (Santa Clara, CA, USA) and USB (Cleveland, OH, USA). Bovine serum albumin (BSA) was obtained from Amresco (Solon, OH, USA) and Sigma. 384-well plates came from BD Biosciences (Franklin Lakes, NJ, USA) and PerkinElmer (Waltham, MA, USA). Kinase-Glo was from Promega (Madison, WI, USA).
10
Primary qHTS Assay for GALK Inhibitors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
EXAMPLE 2
This example demonstrates a primary qHTS assay for inhibitors of GALK.
Assay details and protocol: The primary assay monitored ATP depletion using Promega's KinaseGlo™ technology, where ATP levels are measured through luminescence generated from firefly luciferase, a bioluminescent ATP-dependent enzymes. ATP was held at 35 μM, near its reported KM value, and the KM for galactose was determined under the 1536-well assay conditions to be 50-100 μM (FIG. 1A). As well, the IC50 for a commercially available CD45 inhibitor (N-(9,10-dioxo-9,10-dihydrophenanthren-2-yl)pivalamide) was confirmed (previously found to inhibit GALK (FIG. 1B)). This was used as the positive control for the assay. The assay used 5 nM GALK and a 1 hr incubation time, which gave sufficient signal:background and stability for the HTS. The percent conversion of ATP under these conditions was estimated to be approximately 50% using an ATP standard curve.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!