| Assay Method Information | |
| | Enzyme Activity Assay |
| Description: | The object of this test is to detect the in vitro inhibitory activity of the compound against HER1 (ErbB1), HER2 (ErbB2) and HER4 (ErbB4). The enzymes used in this test were human ErbB1, ErbB2 and ErbB4. Eurofins Pharma Discovery Service provided the activity detection method, and the results of the inhibitory activity of the tested compound against HER1, HER2 and HER4 were shown in Table 1.Experimental Steps and Methods (96-Well Plate):5-fold diluted tested compound buffer (5 μL), peptide substrate poly(Glu, Tyr) (4:1) (2.5 μL), ErbB (4-20 ng, 2.5 μL), MnCl2 (50 mM, 1.25 μL), dH2O (3.75 μL) and [γ-33P]ATP (10 μL) were added, and incubated at 30° C. for 10 min. 3% phosphoric acid was added to terminate the reaction, and 10 μL of the specimen was transferred to Filtermate A. The filter was washed with 75 mM phosphoric acid for three times and methanol once, and transferred to a sealed plastic bag, and a scintillation fluid mixture (4 mL) was added, The intensity of the emitted photons was detected on the scintillation luminescence counter. The photon intensity of the enzyme sample was compared with the photon intensity of the internal control sample, and the level of photon intensity reflected the strength of the tyrosine kinase activity. |
| Affinity data for this assay | |
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