CoralHue - MBLライフサイエンス

1 An MBL Company . Fluorescent Proteins CoralHue .. Amalgaam ( ) . ( . ) ( ) 2001 4 . ( ) Kaede . 15 . Amalgaam ( ) . 2003 1 . CoralHue . CoralHue Coral ( ) Hue ( ) Amalgaam .. monomeric Keima-Red ( ). monomeric Kusabira-Orange ( ). monomeric Azami-Green ( ).. CoralHue 4. Protein Fragment Complementation Method ( ). FRET. FCCS. CoralHue 6. Kaede Kikume Green-Red Dronpa-Green CoralHue 8. Azami-Green Kusabira-Orange Midoriishi-Cyan Keima-Red CoralHue 10. CoralHue . ( ) . Apoptosis .. 12. AMAP Multi Site-directed Mutagenesis Kit ( ). CoralHue . Amalgaam . 17. Fluorescent Proteins CoralHue . Protein Fragment Complementation Method CoralHue .

2 CoralHue (Protein fragment complementation method).. monomeric Kusabira-Green(mKG) 4 .. mKG_N mKG_C A B . A B mKG_N mKG_C mKG . KG .. A. B. mKG_N. mKG_C. NF- B p50 p65 . Fluo-chase Kit . A B . (HEK 293T ). mKG_N mKG_C. A B.. mKG_N mKG_C . Open reading Molecular frame (bp) weight (kDa). mKG. mKG_N 507 mKG_C 156 .. pH . / (M-1cm-1). mKG 494/506 64,200 (494 nm) pKa= 4. TEL: 052-238-1904 FAX: 052-238-1441 Web Site: Fluorescent Proteins FRET Fluorescence resonance energy transfer . CoralHue Midoriishi-Cyan CoralHue Kusabira-Orange Donor (Midoriishi-Cyan) Acceptor (Kusabira-Orange) . Donor .. MiCy mKO . MiCy1 mKO1.

3 EX. EM. EX. EM. 400 450 500 550 600 650. Midoriishi-Cyan(MiCy) Kusabira-Orange( KO) . Wavelength(nm). HeLa 3 . FRET Caspase3 . (3 CCD ASHURA( ) . Caspase 3 +. + 440 nm Caspase3 . (DEVD) . MiCy-DEVD-mKO . Caspase3 . - .. 450 500 550 600 650. MiCy-DEVD-mKO wavelength (nm). ( 440 nm) MiCy DEVD mK0. FCCS Fluorescence Cross Correlation Spectroscopy . CoralHue Azami-Green CoralHue Midoriishi-Cyan . CoralHue Keima-Red 45 nm nm 0. 8. 44. MiCy EM. EX. Keima-Red Midoriishi-Cyan Keima-Red Azami-Green mKeima-Red EM. EX. FCCS 400 500 600 700. Wavelength (nm). I II III. B. Fluo. Int. Fluo. Int. A. B. A Time Time (Fluorescence Cross Correlation Spectroscopy, FCCS) 2.)

4 I A B ( ) . II 2 A B ( ) . III 2 A B ( ) . 5. Web Site . Fluorescent Proteins CoralHue . Kaede Kaede (Trachyphyllia geoffroyi) ( ) (350-410 nm) .. ( ) Kaede . Kikume Green-Red .. 350 410nm . pH . / (M-1cm-1). ( ) 508/518 98,800 pKa= ( ) 572/580 60,400 pKa= . CoralHue Kaede Ex Ex Kaede_Green Em Kaede_Red Em HeLa . Green : EX 475AF20 EM 530DF35. Red : EX 550DF35 EM 575 ALP. DM : 420 DCLP. : Green 400 ms, Red 50 ms 1s 300 400 500 600 700 400 500 600 700. Wavelength(nm) Wavelength(nm). Kikume Green-Red Kikume Green-Red (Favia favas) . 4 ( ) (350-410 nm) .. 37 Kaede .. pH . / (M-1cm-1). ( ) 507/517 53,700 pKa= ( ) 583/593 35,100 pKa= CoralHue KikGR1.

5 Ex Ex . KikGR_Green Em KikGR_Red Em .. 400 500 600 700 400 500 600 700. Wavelength(nm) Wavelength(nm). 6. TEL: 052-238-1904 FAX: 052-238-1441 Web Site: Fluorescent Proteins Dronpa-Green Dronpa-Green (Echinophyllia sp.) . (503 nm) (OFF) 390 nm . (ON) (Photoactivation) . ON / OFF FRAP (Fluorescence Recovery after Photobleaching) FLIP (Fluorescence Loss in Photobleaching) .. pH . / (M-1cm-1). DG1 503/518 95,000 (503 nm) pKa= DG3 491/514 58,000 (487 nm) pKa= CoralHue DG1.. HeLa . Ex Ex EX : 490DF20. DG1 Em DG3 Em EM : 535DF25. DM : 505 DRLPXR. 400 450 500 550 600 400 450 500 550 600. Wavelength (nm) Wavelength (nm).

6 Dronpa-Green1 . 490 nm 400 nm .. pRS316-Arf1p-Dronpa-Green1 . 405 nm .. Arf1p-Dronpa-Green1 YPH499a . 488 nm 405 nm . 2 .. ( ) (Scale bar, 2 m). Olympus FV1000. UPLAPO 60xW multi Ar 488 nm 500-600 nm 405 nm .. 7. Web Site . Fluorescent Proteins CoralHue . Azami-Green Bright Green Azami-Green (Gelaxea fascicularis) 4 (AG) . (mAG1) pH . mAG1 .. pH . / (M-1cm-1). AG 492/505 72,300 (492 nm) pKa< mAG1 492/505 55,500 (492 nm) pKa= . CoralHue mAG1. Ex Ex Em AG Em mAG1 HeLa . U-MGFPHQ (OLYMPUS). EX : BA460-480HQ. EM : BA495-540HQ. DM : DM485. 400 450 500 550 600 400 450 500 550 600. Wavelength(nm) Wavelength(nm).

7 Kusabira-Orange Bright Orange Kusabira-Orange (Fungia concinna) . 2 (KO1) (mKO1 mKO2) . pH mKO1 mKO2 . Midoriishi-Cyan (FRET) .. pH . / (M-1cm-1). KO1 548/561 73,700 (548 nm) pKa< mKO1 548/559 51,600 (548 nm) pKa= mKO2 551/565 63,800 (551 nm) pKa= CoralHue mKO1.. mKO1 HeLa . Ex Ex Em FSET-KOHQ(OLYMPUS). KO1 Em mKO mKO2 EX : 25BP520-540HQ. Ex EM : 25BP555-600HQ. Em DM : DM545HQ. 400 450 500 550 600 650 400 450 500 550 600 650. Wavelength(nm) Wavelength(nm). 8. TEL: 052-238-1904 FAX: 052-238-1441 Web Site: Fluorescent Proteins Midoriishi-Cyan Bright Cyan Midoriishi-Cyan (Acropara sp.) 2 (MiCy1). (mMiCy1).

8 FRET Donor Kusabira-Orange (FRET) .. pH . / (M-1cm-1). MiCy1 472/495 27,250 (472 nm) pKa= mMiCy1 470/496 22,150 (470 nm) pKa= . CoralHue mMiCy1. ( ). Ex Ex MiCy1 Em mMiCy1 Em HeLa . EX : 440AF21. EM : 510WB40. DM : 455 DRLP. 350 400 450 500 550 600 350 400 450 500 550 600. Wavelength(nm) Wavelength(nm). Keima-Red Large Stokes Shift Keima-Red (Montipora sp.) . 2 (dKeima-Red dKeima570) (mKeima-Red) . ( ) . AG MiCy . FCCS(Fluorescence Cross Correlation Spectroscopy) .. pH . / (M-1cm-1). dKeima-Red 440/616 24,600 (440 nm) pKa= dKeima570 440/570 14,000 (440 nm) pKa= mKeima-Red 440/620 14,400 (440 nm) pKa= CoralHue PM-mKeima-Red ( mKeima-Red).

9 DKeima-Red dKeima mKeima-Red Ex Em Ex HeLa . Em dKeima570 EX : 440AF21. Ex Em EM : 610 ALP. Fluo. Int. Fluo. Int. DM : 590 DRLP. 400 500 600 700 400 500 600 700. Wavelength (nm) Wavelength (nm). 9. Web Site . Fluorescent Proteins CoralHue . CoralHue . 24 CoralHue Azami-Green . Kusabira-Orange Kikume Green-Red . ( . ) . humanized Kikume Green-Red1(hKikGR1) . hKikGR1-MC1(AM-V0085) hKikGR1-MCLinker(AM-V0089) -actin HeLa . MC1 hKikGR1 -actin 2 ( ) . MCLinker hKikGR1 -actin 2 . ( ) . hKikGR1/MCLinker hKikGR1 TGNSADGGGGSGGSGGSGGGSTQG GS -actin hKikGR1/MC1 BamHI Not I. hKikGR1 GS -actin BamHI Not I. : BP460-480HQ( ).

10 : BA495-540HQ( ). : DM485(DM). : 546DF20( ). : FF01-624/40-25( ). : FF593-Di02-25x36(DM). UV : BP330-385. : 505 DRLPXR(DM). CoralHue hKikGR1-MC1 CoralHue hKikGR1-MCLinker CoralHue hKikGR1-MCLinker . -actin HeLa -actin -actin . HeLa HeLa . ( ) . ( ) .. pPM-mAG1. pPM-mKO1 . pPM-mKeima-Red pER-mAG1. pER-mKO1. CoralHue pPM-mAG1 CoralHue pPM-mKO1 CoralHue pPM-mKeima-Red .. pNP-AG . pNP-KO1 pMT-mAG1. pMT-mKO1. pMT-mKeima-Red CoralHue pMT-mAG1 CoralHue pMT-mKO1 CoralHue pMT-mKeima-Red .. CoralHue pER-mAG1 CoralHue pER-mKO1 CoralHue pNP-AG CoralHue pNP-KO1. 10. TEL: 052-238-1904 FAX: 052-238-1441 Web Site: Fluorescent Proteins Apoptosis.