PI 3-Kinase HTRF® Assays
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PI 3-Kinase HTRF® Assay kit는 HTRF방식을 이용하여 GRP1 PH domain에 PIP3의 결합이 저해되는 정도를 측정함으로써 테스트 compound의 효과를 분석하는 제품입니다. PI 3-Kinase는 암세포 성장, 당뇨와 관련된 주요 signaling protein으로써, 항암제 개발연구를 위해 사용됩니다. |
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| Eurofins |
- Homogenous time resolved fluorescence (HTRF) 방법을 이용하여 PI 3-Kinase에 대한 potential inhibitor를 스크리닝 합니다.
- PI 3-kinase의 α, β, δ, γ 4가지 isoform에 대해 호환성을 가집니다.
- Z factor가 0.8 이상 일정하므로, 효소반응속도가 안정적이고 믿을 수 있는 결과를 얻을 수 있습니다.
- 기본 방법인 4-step 제품과 보다 더 간편하게 사용할 수 있는 3-step 제품이 제공됩니다.
- 3-step assay kit는 기본 4-step보다 적은 양의 샘플을 사용하며, 분석시간은 줄이고 HTS 분석이 가능합니다.
원리
FRET complex formation
In the absence of PIP3 (e.g. in the absence of kinase, or in the presence of a kinase inhibitor), the FRET complex assembles with the biotinylated PIP3 acting as abridge between the fluorophores. Fluorescence emission is recorded at both 620 nm and 665nm.
FRET complex disruption
The action of PI 3-Kinase on the substrate PIP2 generates the product PIP3, which competes with the biotinylated PIP3 to break up the FRET complex. This leads to an overall decrease in fluorescent signal as kinase activity increases.
종류
4-step(original)과 3-step Assays 비교
| | 4-step | 3-step |
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| Screening throughput |
Low to medium |
Medium to high |
| Recommended plate types |
384-well |
384- and 1536-well |
| Recommended volume |
20 μl |
6 - 20 μl |
| Step |
- Step 1: + Reaction Buffer
(including kinase and PIP2 substrate)
- Step 2: + ATP
(to initiate kinase reaction)
- Step 3: + Stop solutions
(including biotinylated PIP3)
- Step 4: + Detection Mix solutions
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- Step 1: + Reaction Buffer
(including biotinylated PIP3, kinase and PIP2 substrate)
- Step 2: + ATP
(to initiate kinase reaction)
- Step 3: + Stop/Detection Mix Solutions
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Figure 1. Inhibition of PI 3-Kinase isoforms
Protein and lipid kinase inhibitors were tested against four PI 3-Kinase isoforms in a 384-well HTS format to determine % inhibition and identify potent inhibitors.
Figure 2. Comparable data of the 4-step and 3-step assay formats
Titration of the known PI 3-Kinase inhibitor PI-103 demonstrates that the 4-step and 3-step assay formats produce comparable data. Assays were conducted at 100 μM ATP. Data points represent the mean of three replicates and are expressed in terms of the % kinase activity compared to the DMSO control wells.
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