The relative activity of the different PI4KIIIα preparations was assayed as follows. Liposomes containing C16-PI (Echelon Biosciences) were generated by sonication of a 1 mg/mL solution in kinase buffer (100 mM Tris-HCl pH 7.5, 50 mM EGTA, 100 mM MgCl2)) and incubated (at 80 μM) in a 50 μL reaction with 400 ng of PI4KIIIα or PI4KIIIα complex (equal amounts of PI4KIIIα in each sample was ensured by the use of both the BCA assay and SDS-PAGE/Coomassie staining), γ32P-labeled ATP (10 μCi), and cold ATP (50 μM) in kinase buffer for 5 min at 37 °C. The reaction was quenched by the addition of 700 μL of 2:1 chloroform:methanol containing 10 μg/mL Folch fraction (brain phosphoinositides) and 400 μL of 0.1 M HCl. The organic extracts were dried, resuspended in a small amount of 1:1 chloroform:methanol, and equal amounts were analyzed by thin-layer chromatography (mobile phase is 14:32:24:30:64 water:acetic acid:methanol:acetone:chloroform). PI4P was identified by comparison with PI3P generated in a parallel reaction using PI3K p110γ (Sigma) and quantified by autoradiography using a STORM 860 system (Molecular Dynamics).
Pi3k p110γ
Manufactured by Merck Group
PI3K p110γ is a laboratory equipment product that functions as a catalytic subunit of phosphoinositide 3-kinase (PI3K). It plays a role in signal transduction pathways involved in cell growth, proliferation, and survival.
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Lab products found in correlation
2 protocols using pi3k p110γ
1
Quantifying PI4KIIIα Enzymatic Activity
2
Quantifying PI4KIIIα Enzymatic Activity
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The relative activity of the different PI4KIIIα preparations was assayed as follows. Liposomes containing C16-PI (Echelon Biosciences) were generated by sonication of a 1 mg/mL solution in kinase buffer (100 mM Tris-HCl pH 7.5, 50 mM EGTA, 100 mM MgCl2)) and incubated (at 80 μM) in a 50 μL reaction with 400 ng of PI4KIIIα or PI4KIIIα complex (equal amounts of PI4KIIIα in each sample was ensured by the use of both the BCA assay and SDS-PAGE/Coomassie staining), γ32P-labeled ATP (10 μCi), and cold ATP (50 μM) in kinase buffer for 5 min at 37 °C. The reaction was quenched by the addition of 700 μL of 2:1 chloroform:methanol containing 10 μg/mL Folch fraction (brain phosphoinositides) and 400 μL of 0.1 M HCl. The organic extracts were dried, resuspended in a small amount of 1:1 chloroform:methanol, and equal amounts were analyzed by thin-layer chromatography (mobile phase is 14:32:24:30:64 water:acetic acid:methanol:acetone:chloroform). PI4P was identified by comparison with PI3P generated in a parallel reaction using PI3K p110γ (Sigma) and quantified by autoradiography using a STORM 860 system (Molecular Dynamics).
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