Method for measuring kinase activity and kit for measuring
A surfactant and activity technology, applied in the field of kits for measuring kinase activity, can solve the problem of inability to judge receptor kinase and phosphorylation
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Embodiment 1
[0114] Example 1: Detection of substrate phosphorylation by activating HER1
[0115] 1. Preparation method of the sample for reaction
[0116] The cultured cells (MDA-MB468) from breast cancer were placed in 225cm 2 The cells were cultured in the flask until the cells reached 80% confluence (approximately 107 cells). The cultured cells were mixed with 1 ml of cell treatment solution (containing 20 mM HEPES pH7.4, 20 mM MnCl2, 1 mM DTT, 0.2% protease inhibitor (hereinafter referred to as PI), 10% glycerol, 100 μM Na3VO4 and 50 mM NaF). Then, the mixed solution obtained by stirring with a pestle was pressurized to break the cell membrane in the mixed solution to prepare a cell solution. As PI, Protease Inhibitor Cocktail for Mammalian Tissue (manufactured by Sigma Corporation) was used. The obtained cell solution was separated by centrifugation, and the supernatant was discarded. Mix the precipitate and cell membrane solubilizer (containing 20mM HEPES pH 7.4, 20mM MnCl2, 1mM...
Embodiment 2
[0141] Example 2: Detection of Phosphorylated Substrates by Slot Blotting
[0142] Phosphorylated tyrosine was detected by Slot blotting with sample-7 and sample-8 prepared in Example 1.
[0143] There is a slit (slot) imprinter (BIO-DOT SF (Bio-Rad Company)) with a PVDF membrane (Immobilon-P (superscript SQ) transfer membrane 0.2 μm pore size (Millipore (Millipore) Company)) on the base 180 μl of sample-7 and sample-8 were added to each sample well. Sample-7 and Sample-8 were sucked from the back of the PVDF membrane with a vacuum pump, and the protein contained in each sample was adsorbed to the PVDF membrane. The PVDF membrane was removed from the slit blot, shaken in 2 ml of the same primary antibody solution as in Example 1 for 60 minutes, and washed three times with TBS (containing 25 mM Tris and 150 mM NaCl). Then the PVDF membrane was shaken in 2 ml of the same secondary antibody solution as in Example 1 for 60 minutes, washed three times with TBS, dried, and analyze...
Embodiment 3
[0146] Example 3: Effect of HER1 Inhibitors on the Kinase Activity of HER1
[0147] A reaction sample was obtained by the same method as in Example 1, and the effect of the HER1 inhibitor on the kinase activity of HER1 was investigated using the reaction sample.
[0148] 25 µ of the sample for the reaction were put into two test tubes, respectively. A HER1 inhibitor PD168393 (Calbiochem Co.) 200nM reaction solution (containing 20mMPIPES pH7.4, 10mM MnCl2, 1mM DTT, 0.1% NP-40, 0.2% PI, 10% glycerol, 100μM Na3VO4, 20 μM ATP and 3 μg Grb2) 25 μl, another tube was added 25 μl of reaction solution without PD168393, and incubated at 25°C for 30 minutes. The reaction solution containing PD168393 is referred to as PD+, and the reaction solution without PD168393 is referred to as PD-. Western blotting was performed in the same manner as in Example 1 using the PD+ and PD-.
[0149] Growth factor receptor binding protein (Grb2) was constructed as follows. The forward primer (Forwardp...
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