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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
杜建宇 工程师
Page 2
MicroBeta 多功能液体闪烁计数仪
Page 3
MicroBeta 有多种类型
Page 4
MicroBeta 耦合环路设计
Page 5
•Liquid
•Coated plate
•Filter
MicroBeta 接受的样品类型
Page 6
Eppendorf & 4ml 管
45 x 15 mm
Page 7
Rigid or flexible microplates
微孔板
Page 8
滤膜样品
Page 9
Filtermat 滤膜
Page 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 11
Unifilter 滤膜板
Page 12
MicroBeta workstation 软件界面
Page 13
软件介绍—数据显示
Page 14
软件介绍—数据输出
Page 15
MicroBeta 测量模式——按应用分类
Light
LSC SPA
Luminescence Filter plates
Page 16
MicroBeta测量模式的选择
Page 17
Normalization and Standardization
单击“ Protocols” 键 选择“ Normalization” 或“ Standardization”
Page 18
solventbetadecay
fluor light
physical chemical colour
淬灭现象( Quenching )
物理淬灭 化学淬灭 颜色淬灭
Page 19
0
20
40
60
80
100
120
200 250 300 350 400
SQP(I)
C-1
4 E
ffic
ien
cy /
%
淬灭曲线( Quench Curve )
Page 20
OH
ATP
ADP
O
PO
OH
OH
蛋白激酶
Page 21
蛋白激酶的细胞模式
Page 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 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, 6005177) 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)
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+
Scintillation Proximity Assay
Page 25
+Light
Scintillation Proximity Assay
Page 26
White plastic
Coating ofscintillator
Flashplate 微孔板
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FlashPlate Scintillation Proximity Assays for Characterization and Screening of DNA Polymerase, Primase, and Helicase Activities
J Biomol Screen.2001; 6: 39-46
FlashPlate Scintillation Proximity Assays
Page 28
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).
Flashplate 应用—— MAP Kinase
Page 29
发光检测( Luminescence )
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 30
Any Questions?
Thank you!