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

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MicroBeta 多功能液体闪烁计数仪

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MicroBeta 有多种类型

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MicroBeta 耦合环路设计

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•Liquid

•Coated plate

•Filter

MicroBeta 接受的样品类型

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Eppendorf & 4ml 管

45 x 15 mm

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Rigid or flexible microplates

微孔板

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滤膜样品

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Filtermat 滤膜

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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

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Unifilter 滤膜板

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MicroBeta workstation 软件界面

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软件介绍—数据显示

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软件介绍—数据输出

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MicroBeta 测量模式——按应用分类

Light

LSC SPA

Luminescence Filter plates

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MicroBeta测量模式的选择

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Normalization and Standardization

单击“ Protocols” 键 选择“ Normalization” 或“ Standardization”

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solventbetadecay

fluor light

physical chemical colour

淬灭现象（ Quenching ）

物理淬灭 化学淬灭 颜色淬灭

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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 ）

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OH

ATP

ADP

O

PO

OH

OH

蛋白激酶

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蛋白激酶的细胞模式

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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

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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

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+Light

Scintillation Proximity Assay

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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

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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

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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

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Any Questions?

Thank you!