The kinase activity of PI3K alpha was measured by using the commercial ADP Hunter™ Plus assay (DiscoveRx), a homogeneous assay measuring ADP accumulation, as a universal product of kinase activity. The assay was done following general manufacturer recommendations and adapting protein and substrate concentrations to optimal conditions. Kinase buffer was 50 mM HEPES, pH 7.5, 100 mM NaCl, 1 mM EGTA, 0.04% CHAPS, 3 mM MgCl2, and 10 μg/ml BGG (bovine γ‐globulin), 2 mM TCEP. The assay was done at 100 μM PIP2, as peptide substrate, and 50 μM ATP. Protein concentration was 0.8 ng/μl. In order to calculate the IC50 of described compounds, serial 1:5 dilutions were prepared, and the reaction started by the addition of ATP. Incubation was done for 1 h at 25°C. Reagents A and B (DiscoveRx) were sequentially added to the wells, and plates were incubated for 30 min at 37°C. Fluorescence counts were read in a Victor instrument (PerkinElmer) with the recommended settings (544 and 580 nm as excitation and emission wavelengths, respectively). Values were plotted against inhibitor concentration and fit to a sigmoid dose–response curve with the GraphPad software.
Victor instrument
Manufactured by PerkinElmer
Sourced in Finland
The Victor instrument is a multi-mode microplate reader designed for a variety of applications in life science research and drug discovery. It is capable of performing absorbance, fluorescence, and luminescence measurements.
Automatically generated - may contain errors
7 protocols using victor instrument
1
Kinase Activity Assay for PI3K alpha
2
Kinase Activity Measurement Protocols
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PIM1/3 kinase activities were measured as previously described in Martínez-Gonzalez et al. [49 (link)]. HASPIN activity was measured using the commercial ADP GLO assay (Promega), a homogeneous assay measuring ADP accumulation, as a universal product of kinase activity. The assay was done following general manufacturer recommendations and adapting protein to optimal conditions. Kinase buffer was 15 mM HEPES ((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), pH 7.4, 20 mM NaCl, 1 mM egtazic acid (EGTA), 0.02% Tween-20, 10 mM MgCl2 and 0.1 mg/mL BGG (bovine γ-globulin). The assay to measure autophosphorylation of HASPIN (ProQinase, Freiburg, Germany) was done at 150 μM ATP. In order to calculate the IC50 of described compounds, serial 1:5 dilutions were tested. Luminescence counts were read in a Victor instrument (Perkin Elmer) Values were plotted against inhibitor concentration and fit to a sigmoid dose–response curve with activity base from IDBS software. TYK2 kinase assay was performed at ProQinase.
3
Measuring PI3K Kinase Inhibition
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The kinase activity was measured by using the commercial ADP HunterTM Plus assay available from DiscoveRx (#33-016), which is a homogeneous assay to measure the accumulation of ADP, a universal product of kinase activity. The enzyme, PI3K (p110α) was purchased from Cama Biosciences (#07CBS-0402A). The assay was performed following the manufacturer recommendations. To calculate the IC50 of the selected compounds, serial 1:5 dilutions were used in a concentration range from 128 pM to 50 μM fluorescence counts were read in a Victor instrument (Perkin Elmer) with the recommended settings (544 and 580 nm as excitation and emission wavelengths, respectively). Values were normalized against the control activity included for the enzyme (Le., 100 % PI3 kinase activity, without compound). These values were plotted against the inhibitor concentration and were fit to a sigmoid dose-response curve by using Activity base software from IDBS.
4
Evaluating Anti-Cancer Efficacy of Tyrosine Kinase Inhibitors
5
PIM Kinase Activity Assay Protocol
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The PIM kinase activities were measured using the commercial ADP Hunter™ Plus assay (DiscoveRx), a homogeneous assay measuring ADP accumulation, as a universal product of kinase activity. The assay was done following general manufacturer recommendations and adapting protein and substrate concentrations to optimal conditions. Kinase buffer was 15 mM HEPES, pH 7.4, 20 mM NaCl, 1 mM EGTA, 0.02% Tween‐20, 10 mM MgCl2, and 0.1 mg/ml LBGG (bovine γ‐globulin). All PIM kinase assays were done at 100 μM PIMtide (ARKRRRHPSGPPTA), as peptide substrate, and 100 μM ATP. Protein concentration was 50, 350, and 500 ng/μl for PIM1, PIM2, and PIM3, respectively. In order to calculate the IC50 of described compounds, serial 1:5 dilutions were prepared, and the reaction started by the addition of ATP. Incubation was done for 1 h at 25°C. Reagents A and B (DiscoveRx) were sequentially added to the wells, and plates were incubated for 30 min at 37°C. Fluorescence counts were read in a Victor instrument (PerkinElmer) with the recommended settings (544 and 580 nm as excitation and emission wavelengths, respectively). Values were plotted against inhibitor concentration and fit to a sigmoid dose–response curve with the GraphPad software.
6
Cytotoxicity Evaluation of Peptides Using MTT Assay
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Immunoassay Measurement of Prostate Biomarkers
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