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放射性同位素检测平台——MicroBeta 及其在蛋白激酶筛选中的应用

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Presentation on theme: "放射性同位素检测平台——MicroBeta 及其在蛋白激酶筛选中的应用"— Presentation transcript:

1 放射性同位素检测平台——MicroBeta 及其在蛋白激酶筛选中的应用
杜建宇 工程师

2 MicroBeta多功能液体闪烁计数仪 Page 2

3 MicroBeta 有多种类型 Page 3

4 MicroBeta 耦合环路设计 Page 4

5 MicroBeta 接受的样品类型 Liquid Coated plate Filter Page 5

6 Eppendorf & 4ml 管 45 x 15 mm Page 6

7 Rigid or flexible microplates
微孔板 Rigid or flexible microplates Page 7

8 滤膜样品 Page 8

9 Filtermat 滤膜 Page 9

10 FilterMate 滤膜样品收集系统 Waste bottles and stands
Pressurized wash bottle assembly UniFilter plate in position for harvesting Holder for self-aligning and plain, non-self-aligning filter mats Page 10

11 Unifilter 滤膜板 Page 11

12 MicroBeta workstation 软件界面
Page 12

13 软件介绍—数据显示 Page 13

14 软件介绍—数据输出 Page 14

15 MicroBeta测量模式——按应用分类
LSC SPA Luminescence Filter plates Page 15

16 MicroBeta测量模式的选择 Page 16

17 Normalization and Standardization
单击“Protocols”键 选择“Normalization”或“Standardization” Page 17

18 beta decay solvent fluor light physical chemical colour
淬灭现象(Quenching) beta decay solvent fluor light physical chemical colour 物理淬灭 化学淬灭 颜色淬灭 Page 18

19 淬灭曲线(Quench Curve) Page 19

20 蛋白激酶 Page 20

21 蛋白激酶的细胞模式 Page 21

22 滤膜应用——激酶实验 BIBW2992, an irreversible EGFR/HER2 inhibitor highly effective in preclinical lung cancer models Oncogene, April 14, 2008 Page 22

23 激酶实验——33P[ATP] In vitro kinase activity assay For VEGF2 and HGFR kinase assays, a random polymer pEY (4:1) from SIGMA was used as substrate. Each 50 µl-reaction contained 5 % Me2SO, 40 mM HEPES pH 7.4, 5 mM MgCl2 0.5 mg/ml pEY, 0.05% Triton X-100, 100 µM ATP, 1 µCi [γ 33P] ATP and 10 µl of enzyme preparation. … … The precipitate was then trapped onto GF/B Unifilters (Packard, ) using a 96 well filter mate universal harvester (Packard, C961961). … … radioactivity was determined by scintillation counting using a TopCount™ (Packard) or a Microbeta b counter™ (Wallac) Page 23

24 Scintillation Proximity Assay
Page 24

25 Scintillation Proximity Assay
Page 25

26 Flashplate微孔板 Coating of scintillator White plastic Page 26

27 FlashPlate Scintillation Proximity Assays
FlashPlate Scintillation Proximity Assays for Characterization and Screening of DNA Polymerase, Primase, and Helicase Activities J Biomol Screen.2001; 6: 39-46 Page 27

28 Flashplate应用——MAP Kinase
MAP (mitogen activated protein) kinase activity can be measured through the use of MBP (myelin basic protein) as a substrate in the presence of appropriate buffer and [33P]-g-ATP. Activated MAP kinase incorporates the 33P onto the serine and threonine residues of MBP. When increasing amounts of MBP are coated onto FlashPlate, counts bound increase without increasing background (Fig. 1). Page 28

29 X 发光检测(Luminescence) …have optimised luminescence performance
Single photon event Easier to remove background High speed electronics required up to 20 x 106 CPS Temperature control option for MicroBeta X …have optimised luminescence performance Page 29

30 Any Questions? Thank you! Page 30

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