Recombinant kinase inhibition was measured using standard radioassay. Bcr-Abl1 and Cdk-2/cyclin E kinases were produced in-house, FLT3-ITD and BTK were purchased from ProQinase. The kinases were assayed with suitable substrates (500 µM peptide GGEAIYAAPFKK for Abl, 1 mg/mL histone H1 for Cdk-2, 100 µM poly (Glu:Tyr; 4:1)) in the presence of ATP (1 µM for Bcr-Abl, 15 µM for Cdk-2 and BTK), 0.05 µCi [γ-33P]ATP and the test compound in reaction buffer (60 mM HEPES-NaOH, pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 3 µM Na orthovanadate, 1.2 mM DTT, 2.5 µg/50 µL PEG20.000). The reactions were stopped by adding 3% aq. H3PO4. P-81 phosphocellulose (Whatman) was used to separate phosphorylated substrates. Kinase activity was quantified using a FLA-7000 digital image analyser. The concentration of the test compounds required to reduce the activity by 50% (IC50 value) was determined from dose–response curves.
P 81 phosphocellulose
Manufactured by Cytiva
Sourced in United Kingdom
The P-81 phosphocellulose is a laboratory filtration and chromatography product used for the separation and purification of biological molecules, such as proteins and nucleic acids. It is made of phosphorylated cellulose, which provides a stable and consistent ion exchange matrix for the selective retention and elution of target analytes.
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Lab products found in correlation
3 protocols using p 81 phosphocellulose
1
Recombinant Kinase Inhibition Assay
2
Abl1 Kinase Inhibition Assay
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Inhibitory activity of prepared compounds was tested on a recombinant Abl1 kinase produced in-house. The kinase was assayed with a substrate 500 µM peptide GGEAIYAAPFKK in the presence of 1 µM ATP + [γ-33P]ATP (0.05 µCi per reaction) and the test compound in a final volume of 10 µL of reaction buffer (60 mM HEPES-NaOH, pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 3 μM Na-orthovanadate, 1.2 mM DTT, 2.5 μg/50 μL PEG20.000). The reactions were stopped by adding 5 µL of 3% aq. H3PO4. Aliquots were spotted onto P-81 phosphocellulose (Whatman, Maidstone, UK), washed 3× with 0.5% aq. H3PO4, and air-dried. Peptide phosphorylation was quantified using digital autoradiography (FLA-7000, Fujifilm, Tokyo, Japan). The concentration of the test compounds required to reduce the activity by 50% was determined from dose–response curves and reported as the IC50 value.
3
CDK2/Cyclin E Kinase Inhibition Assay
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The tested compounds were dissolved in DMSO and diluted with water (the concentration of DMSO in the reaction never exceeded 0.2%). The CDK2/Cyclin E complex was produced in Sf9 insect cells via baculoviral infection and purified on a Ni 2+ NTA column (Qiagen). Kinase (approx. 10 ng) was assayed using a mixture of the following: 1 mg/mL of histone H1, 15 µM of ATP, 0.05 of µCi [γ-33P]ATP, the tested compound, and reaction buffer, in a final volume of 10 µL. The reaction buffer consisted of: 60 mM of HEPES-NaOH, pH 7.5, 3 mM of MgCl2, 3 mM of MnCl2, 3 μM of Na-orthovanadate, 1.2 mM of DTT, and 2.5 μg / 50 μl of PEG20.000.
The reactions were stopped by adding 5 µL of 3% aqueous H3PO4. Aliquots were spotted onto P-81 phosphocellulose (Whatman), washed 3 times with 0.5% aqueous H3PO4, and finally airdried. Kinase inhibition was quantified using a FLA-7000 digital image analyzer (Fujifilm).
The concentration of each tested compound required the decrease of the CDK activity by 50%.
The IC50 values were determined from the dose-response curve.
The reactions were stopped by adding 5 µL of 3% aqueous H3PO4. Aliquots were spotted onto P-81 phosphocellulose (Whatman), washed 3 times with 0.5% aqueous H3PO4, and finally airdried. Kinase inhibition was quantified using a FLA-7000 digital image analyzer (Fujifilm).
The concentration of each tested compound required the decrease of the CDK activity by 50%.
The IC50 values were determined from the dose-response curve.
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