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Home > ÀüÁ¦Ç°º¸±â > Çü±¤ ´Ü¹éÁú ¡¤ Reporter > Reporter Systems > Live Cell Reporter Assay ºÐºñÇü Reporter (Metridia Luciferase)

Live Cell Reporter Assay ºÐºñÇü Reporter (Metridia Luciferase)

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Á¦Á¶»ç Á¦Ç°ÄÚµå Á¦Ç°¸í ¿ë·® °¡°Ý
(ºÎ°¡¼¼º°µµ)
ºñ°í »ç¿ëÀڸŴº¾ó
Clontech
631726
Ready-To-Glow Secreted Luciferase Reporter Assay
°ü·ÃÇмú ±¸¸ÅÇϱâ
100 ȸ
212,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â x32993
Clontech
631727
Ready-To-Glow Secreted Luciferase Reporter Assay
°ü·ÃÇмú ±¸¸ÅÇϱâ
500 ȸ
731,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â
Clontech
631728
Ready-To-Glow Secreted Luciferase Reporter Assay
°ü·ÃÇмú ±¸¸ÅÇϱâ
1,000 ȸ
1,028,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â
Clontech
631734
Ready-To-Glow Dual Secreted Reporter Assay
°ü·ÃÇмú ±¸¸ÅÇϱâ
500 ȸ
1,688,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â x32992
Clontech
631740
Ready-To-Glow Automation Kit
°ü·ÃÇмú ±¸¸ÅÇϱâ
5,000 ȸ
3,733,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â x32991, x32994
Clontech
631781
Ready-To-Glow MetLuc Monoclonal Antibody
°ü·ÃÇмú ±¸¸ÅÇϱâ
50 ug
1,075,000¿ø  Á¦Á¶»ç ÆäÀÌÁö·Î ¹Ù·Î°¡±â

  • °£´ÜÇÏ°í ¹Î°¨ÇÏ°Ô promoter È°¼ºÀ» °üÂû
  • °­·ÂÇÏ°í ¾ÈÁ¤ÀûÀÎ signal
  • »ì¾Æ ÀÖ´Â ¼¼Æ÷ ºÐ¼® - time course ½ÇÇè¿¡ ÀûÇÕ
  • High-throughput ½ÇÇè¿¡ ÃÖÀû
ClontechÀÇ live cell secreted reporter´Â ´Ù¸¥ transcriptional reporter ÀÌ»óÀ¸·Î ¸¹Àº ÀåÁ¡À» °®°í ÀÖ´Ù. À̵éÀº ¹è¾ç ¹èÁö·Î ºÐºñµÇ±â ¶§¹®¿¡ ¹Ýº¹ÀûÀ¸·Î °°Àº ¹è¾ç¾×À» ½Ã·á·Î ÇÏ¿© À¯ÀüÀÚ ¹ßÇö¿¡ ´ëÇÑ kinetics¸¦ ÃøÁ¤ÇÒ ¼ö ÀÖ´Ù. ¶ÇÇÑ Northern blot, RNase protection assay, Western blot°ú °°Àº ¹æ¹ýÀ» »ç¿ëÇÏ¿© °°Àº ¼¼Æ÷·Î Ãß°¡ÀûÀÎ ¿¬±¸¸¦ ÇÒ ¼ö ÀÖÀ¸¸ç, 96-well ¿¡¼­ 1,536-well plateÀÇ ¹üÀ§±îÁö ºÐ¼®ÇÒ ¼ö ÀÖ´Ù.
Ready-To-Glow Live Cell Secreted Reporter System
Ready-To-Glow SystemÀº secreted Metridia luciferase reporter¿¡ ±â¹ÝÀ» µÎ°í ÀÖÀ¸¸ç, enzyme-based systemÀÇ ¹Î°¨¼º°ú live-cell assayÀÇ ÀÌÁ¡À» °áÇÕ½ÃŲ Á¦Ç°ÀÌ´Ù. ¼¼Æ÷¸¦ ÆļâÇÏÁö ¾Ê°í transfection µÈ ¼¼Æ÷ÀÇ »óÃþ¾×À¸·Î ºÐºñµÈ reporter È¿¼ÒÀÇ È°¼ºÀ» one-stepÀ¸·Î ÃøÁ¤ÇÔÀ¸·Î½á promoter È°¼ºÀ» °üÂûÇÒ ¼ö ÀÖ´Ù (Figure 1).

Firefly¿Í Renilla luciferase´Â cytosol proteinÀ̱⠶§¹®¿¡ reporter¸¦ °ËÃâÇϱâ À§Çؼ­´Â ¹Ýµå½Ã ¼¼Æ÷¸¦ ÆļâÇÏ°í ±âÁúÀ» ÷°¡ÇÏ¿©¾ß ÇÑ´Ù. µû¶ó¼­, timecourse ¿¬±¸¿¡¼­´Â transfection µÈ ¼¼Æ÷¿¡¼­ ÀÇ¹Ì ÀÖ´Â °á°ú¸¦ ¾ò±â À§Çؼ± ½Ã°£ º°·Î ¼¼Æ÷¸¦ ÆļâÇؾ߸¸ ÇÏ´Â ºÒÆíÇÔÀÌ ÀÖ´Ù. ±×·¯³ª Ready-to-Glow Luciferase SystemÀº ÀÌ·¯ÇÑ Àå¾Ö ¿ä¼ÒÀ» Á¦°Å½ÃÄ×´Ù.

Metridia secreted luciferase´Â Renilla¿Í firefly luciferase¿Í °°ÀÌ ºÐºñµÇÁö ¾Ê´Â luciferase reporter¿Í ºñ±³ÇÏ¿© ±âÁú ÷°¡ ÈÄ signal °­µµÀÇ º¯È­ ¾øÀÌ ´õ ³ôÀº signal ¾ÈÁ¤¼ºÀ» º¸¿´´Ù (Figure 2). ÀÌ°ÍÀº Çѹø¿¡ ´Ù¼öÀÇ ½Ã·á¸¦ ´Ù·ç±â ½±µµ·Ï ÇØÁØ´Ù. ¶ÇÇÑ signal intensity°¡ ±âÁú ÷°¡ ÈÄ ½Ã°£¿¡ µû¶ó °¨¼ÒÇÏ´õ¶óµµ, ÀüüÀûÀÎ ¹ßÇö ¹è¼ö´Â 30 ºÐ ÈÄ¿¡µµ °°Àº »óÅ·Π³²¾ÆÀÖ°Ô µÈ´Ù(Figure 3).

ÀçÁ¶ÇÕ Metridia luciferase È°¼ºÀº 96-well ÇüÅ (40 fg Metridia luciferase per ml of sample)¿¡¼­ ¸Å¿ì ³·Àº °ËÃâ ÇÑ°è (~2 fg per well)¸¦ °¡Áö¸ç, ¾ÆÁÖ ³ÐÀº ¹üÀ§ÀÇ ³óµµ (Àû¾îµµ 6 log)¿¡¼­µµ Á÷¼±ÀûÀÎ °á°ú¸¦ ³ªÅ¸³½´Ù. ÀÌ È¿¼ÒÀÇ dynamic rage´Â ´Ù¼öÀÇ ¼¼Æ÷ Á¾·ù¿Í plate ÇüÅ¿¡¼­ ÃøÁ¤ÀÌ µÇ¾úÀ¸¸ç, Èñ¼®µÈ ¹èÁö »óÃþ¾×¿¡¼­ signalÀº Àû¾îµµ 4 ÀÚ¸® ¼ö (0.01% ~ 100% ; 2)¿¡ ´ëÇÏ¿© ¼±ÇüÀ» ³ªÅ¸³½´Ù. Z¡¢°ªÀº 0.66À̸ç, ÀÌ°ÍÀº ³·Àº °¡º¯¼º°ú ÇÔ²² ³ôÀº dynamic range¸¦ ´ëº¯ÇØ ÁØ´Ù. Metridia luciferase´Â 2% DMSO¿¡¼­µµ ¾ÈÁ¤ÇÏ´Ù. Hight throughput applicationÀ» À§ÇÑ ´õ ¸¹Àº Á¤º¸°¡ ÇÊ¿äÇÏ´Ù¸é Clontech website¿¡¼­ È®ÀÎÇÒ ¼ö ÀÖ´Ù.


Figure 1. Flow chart of the Ready-To-Glow Secreted Luciferase Reporter System.


Figure 2. High signal intensity and stability using secreted Metridia luciferase. CHO cells were plated into 96-well plates and transiently transfected with CMV-driven constructs encoding non-secreted firefly luciferase, non-secreted Renilla luciferase, and secreted Metridia luciferase. 24 hr after transfection, luciferase activity in equivalent samples was analyzed by addition of the recommended substrate. The signal was measured at different timepoints over a period of 45 min. neg = negative control.


Figure 3. Monitoring promoter activation using the sequence-optimized secreted Metridia luciferase reporter. HeLa cells were transiently transfected with a vector construct containing the NF¥êB response element driving the expression of sequence-optimized secreted Metridia luciferase. 24 hr after transfection, the media was removed and replaced by media with or without TNF-¥á (100 ng/ml) to activate the NF¥êB signal transduction pathway. Six hr after addition of TNF-¥á, samples of the media were removed and analyzed for Metridia luciferase activity. The fold induction was calculated for different time points following the addition of substrate.
Ready-To-Glow Dual Secreted Reporter System
Ready-To-Glow Dual Secreted Reporter SystemÀº Ready-To-Glow Secre ted Luciferase¿Í secreted alkaline phosphatase (SEAP)ÀÇ 2 Á¾ÀÇ ºÐºñÇü report·Î ±¸¼ºµÇ¾î ÀÖÀ¸¸ç, reporter-specific substrate¸¦ ÀÌ¿ëÇÏ¿© ÃøÁ¤ÇÏ°í ±¸º°ÇÒ ¼ö ÀÖ´Ù. µÎ reporterÀÇ secretion kineticsÀº ¸Å¿ì À¯»ç ÇÏ¿© (Figure 4), ¼¼Æ÷ ¹è¾ç ¹èÁö¿¡¼­ ÃøÁ¤µÇ´Â ÇÑ reporterÀÇ ¾çÀº promoter È°¼ºÀ» Á¤È®ÇÏ°Ô ¹Ý¿µÇÑ´Ù. ÀÌ·¯ÇÑ dual-reporter ½Ã½ºÅÛÀº ´Ù¾çÇÑ ÇüÅ·ΠµÎ promoterÀÇ È°¼º º¯È­¸¦ °üÂûÇϵµ·Ï ÇØÁØ´Ù (Figure 5). ÇϳªÀÇ reporter´Â transfection È¿À²À» ÃøÁ¤Çϱâ À§ÇÑ control·Î »ç¿ëÇÏ°í, ´Ù¸¥ Çϳª´Â ¸ñÀû promoter¸¦ °üÂûÇϱâ À§ÇØ »ç¿ëÇÒ ¼ö ÀÖ´Ù.
Great EscAPe SEAP Reporter System
Secreted Alkaline Phosphatase (SEAP)Àº transfectionµÈ ¼¼Æ÷¿¡¼­ RNA ¹ßÇö ¼öÁØ¿¡ ºñ·ÊÇÏ¿© ¹è¾ç ¹èÁö·Î ºÐºñµÈ´Ù (Figure 5). SEAP assay´Â È¿¼Ò ³óµµÀÇ 104 ¹è ¹üÀ§±îÁö ¼±ÇüÀ» ÀÌ·é´Ù. ClontechÀº chemiluminescent¿Í fluorescent substrate¸¦ ¸ðµÎ Á¦°øÇϸç, chemiluminescent assay´Â SEAP ´Ü¹éÁú 10-13 g ±îÁöµµ °ËÃâÇÒ ¼ö ÀÖ´Ù. Fluorescent assay´Â firefly luciferase assay º¸´Ù ´ú ¹Î°¨ÇÏÁö¸¸, ´ëºÎºÐÀÇ ½ÇÇè¿¡ ÀûÇÕÇÏ´Ù.


Figure 4. Similar secretion kinetics of Metridia secreted luciferase and SEAP enable accurate comparisons of promoter activity. HeLa cells were transiently transfected with either pMetLuc-Control or pSEAP-Control in six-well plates. Media samples were collected at each time point and each sample was tested in triplicate.


Figure 5. Monitoring activation of two promoters simultaneously. HEK 293 cells cotransfected with pNF¥êB-TA-MetLuc and pCRE-SEAP constructs were treated with fresh media alone or with media containing either 1,000 ng/ml TNF-¥á or 10 ¥ìM forskolin. Samples of culture supernatant were collected and assayed 7 hr later.
Components & Storage Conditions
°¢ Á¦Ç° ±¸¼º¹°°ú º¸°üÁ¶°ÇÀº Certificate of Analysis ¸¦ ÂüÁ¶ÇϽʽÿÀ.

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