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 Á¦Ç°¾È³»  ºÐÀÚ»ý¹°ÇР  Protein Research  Protein Kinase Research

PI 3-Kinase HTRF¢ç Assays

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.

Eurofins
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  • 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 formation
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.
FRET complex formation
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4-step(original)°ú 3-step Assays ºñ±³
 4-step3-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
  • 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.
Inhibition of PI 3-Kinase isoforms
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.
Comparable data of the 4-step and 3-step assay formats
Comparable data of the 4-step and 3-step assay formats
ÁÖ¹®Á¤º¸(Ordering Information)
CATNUM PRODUCT SIZE
33-036PI3 Kinase HTRF Assay - 10,000 wellsEA
33-017PI3 Kinase HTRF Assay - 1920 wellsEA
33-016PI3 Kinase HTRF Assay - 384 wellsEA
33-037PI3 Kinase HTRF Assay - 50,000 wellsEA
33-039PI3-K (CII) HTRF Assay - 5-plate kitEA
33-038PI3-K (CII) HTRF Assay 1-plate kitEA
33-047PI3-K 3-step HTRF assay kit, 10K wellsEA
33-041PI3-K 3-step HTRF assay kit, 1920 wellsEA
33-040PI3-K 3-step HTRF assay kit, 384 wellsEA
33-055PIP4-Kinase HTRF Assay (1920 wells)EA
33-054PIP4-Kinase HTRF Assay (384 wells)EA
33-126PIP5-Kinase HTRF Assay (1920 wells)EA
33-125PIP5-Kinase HTRF Assay (384 wells)EA