PI 3-Kinase HTRF¢ç Assays
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PI 3-Kinase HTRF¢ç Assay kit´Â HTRF¹æ½ÄÀ» ÀÌ¿ëÇÏ¿© GRP1 PH domain¿¡ PIP3ÀÇ °áÇÕÀÌ ÀúÇØµÇ´Â Á¤µµ¸¦ ÃøÁ¤ÇÔÀ¸·Î½á Å×½ºÆ® compoundÀÇ È¿°ú¸¦ ºÐ¼®ÇÏ´Â Á¦Ç°ÀÔ´Ï´Ù. PI 3-Kinase´Â ¾Ï¼¼Æ÷ ¼ºÀå, ´ç´¢¿Í °ü·ÃµÈ ÁÖ¿ä signaling proteinÀ¸·Î½á, Ç×¾ÏÁ¦ °³¹ß¿¬±¸¸¦ À§ÇØ »ç¿ëµË´Ï´Ù. |
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Eurofins |
- Homogenous time resolved fluorescence (HTRF) ¹æ¹ýÀ» ÀÌ¿ëÇÏ¿© PI 3-Kinase¿¡ ´ëÇÑ potential inhibitor¸¦ ½ºÅ©¸®´× ÇÕ´Ï´Ù.
- PI 3-kinaseÀÇ α, β, δ, γ 4°¡Áö isoform¿¡ ´ëÇØ ȣȯ¼ºÀ» °¡Áý´Ï´Ù.
- Z factor°¡ 0.8 ÀÌ»ó ÀÏÁ¤ÇϹǷÎ, È¿¼Ò¹ÝÀÀ¼Óµµ°¡ ¾ÈÁ¤ÀûÀÌ°í ¹ÏÀ» ¼ö ÀÖ´Â °á°ú¸¦ ¾òÀ» ¼ö ÀÖ½À´Ï´Ù.
- ±âº» ¹æ¹ýÀÎ 4-step Á¦Ç°°ú º¸´Ù ´õ °£ÆíÇÏ°Ô »ç¿ëÇÒ ¼ö ÀÖ´Â 3-step Á¦Ç°ÀÌ Á¦°øµË´Ï´Ù.
- 3-step assay kit´Â ±âº» 4-stepº¸´Ù ÀûÀº ¾çÀÇ »ùÇÃÀ» »ç¿ëÇϸç, ºÐ¼®½Ã°£Àº ÁÙÀ̰í HTS ºÐ¼®ÀÌ °¡´ÉÇÕ´Ï´Ù.
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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.

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4-step(original)°ú 3-step Assays ºñ±³
| 4-step | 3-step |
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.

