ab128503 PI3 Kinase Assay Kit - Abcam

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5. Assay ProcedureA. Kinase Assay1. To prepare the Kinase Assay, mix the following componentsfor each assay point in separate vials:Reaction Buffer 25 µlLipid Substrate Vesicle 10 µlPI 3-Kinase Sample5 µl(diluted as appropriate)2. Incubate 5 min at room temperature3. Add 10 µl [γ 32 P]-ATP Mix to start the reaction4. Incubate 15 min at 37°C5. Stop the reaction by addition of 100 µl HCl (1 M) and vortexB. Lipid Extraction1. Add 300 µl of chloroform / methanol (1:1)2. Vortex the mix and centrifuge for 1 min to separate thephases3. Remove the upper aqueous phase4. Wash the organic layer with 400 µl HCl (1 M) and removethe water phase7
C. Quantification1. Transfer 100 µl of the organic phase onto a silica gel TLCplate and run it in 2 M acetic acid / isopropanol (1:2)2. Expose the plate for visualization and quantification ofPI3 Kinase spots in a scintillation counter3. Alternatively, a 100 µl aliquot of the washed chloroformphase may be counted by liquid scintillationFor further technical questions please do not hesitate tocontact us by email (technical@abcam.com) or phone(select “contact us” on www.abcam.com for the phonenumber for your region).8
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Page 4 and 5: 1. IntroductionPhosphoinositide 3-k
Page 6 and 7: 3. Components and StorageA. Kit Con
Page 12: UK, EU and ROWEmail: technical@abca

ab128503 PI3 Kinase Assay Kit - Abcam

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