緑色蛍光蛋白（eGFP）の検出 Tecan Ultra Evolution …

1 1 eGFP Tecan Ultra Evolution Safire GENios Pro GFP pacific jellyfish Renilla reniformis GFP cDNA GFP GFP GFP N- C- GFP - 238 / 395 nm 475 nm 30,000 7000 M-1cm-1 70 80% 508 nm GFP Ser-Tyr-Gly 4-(p- ) -5-1 2 GFP GFP 90 C 6 M pH GFP GFP 1 2 GFP 3 1 GFP A431 25000 / / Greiner Costar eGFP A431 ATCC No.

2 CRL-1555 Barbara Krammer L- Na-Pyruvat / HEPES 5% FCS, PAA laboratories, Austria DMEM Sigma, D-5671 CO2 37 C Lipofectin Reagent 18292-011, Invitrogen pEGFP-N1 Cat. No.: #6085-1 Clonetech GFP GFP 96- Costar Szabo-Scandic, Austria, Cat. No.: 3603 Greiner, Cat. 96 TC 25000 / / 1 COSTAR GREINER 96 50000 / 2 97 / 50000 1 / / 1 0 50000 2 50000 0 3 25000 25000 4 12500 37500 5 6250 43750 6 3125 46875 7 1563 48437 8 768 49232

3 9 384 49616 10 192 49808 11 97 49903 12 0 0 1 GFP 1 50000 Tecan Ultra Evolution Tecan GENios Pro Tecan Safire GFP Ex 485(20)/Em 535(25) Ex 465(35)/Em 515(20) Ex 485(20)/Em 515(20) Ex 485(20)/Em 520(20) 3 / 320 500 nm 520 800 nm 505 nm Ultra Evolution GENios Pro 1 1 Z- 3 DME Sigma, Cat.

4 No. 5030 GFP Tecan Safire eGFP Hoechst 33342 Hoechst 33342 minor groove-binding DNA DNA Molecular Probes: H-1399, H-3570 Hoechst 33342 PBS 55 g/ml DME Sigma, Cat. No. 5030 10 mM Hepes 4 mM L- 1 mM Na-Pyruvat 100 l 10 l CO2 37 C 1 GFP 0 1 400 425 450475 500525550 nm A. U. 2 eGFP Excitation spectrum of GFP GFP GFP GFP eGFP- 1 Spot II CCD Diagnostic Instrument Olympus IX70 600 Zeiss Filter set 17: BP 485(20), FT 510, BP 515-565 GFP 2 490 nm 510 nm 1 Ex 485(20)/Em 535(25) 2 Ex 465(35)/Em 515(20) 3 Ex 485 (20)/Em 515(20) 4 Ex 485(20)/Em 520(20) Tecan Ultra Evolution GENios Pro Safire 12 nm 1 1 1 mrpw 3 3 1 4 4 25000 / 25000 / Greiner Costar Ex 485(20)/Em 535(25) Ex 465(35)/Em 515(20) Ex 485 (20)/Em 515(20) Ex 485(20)/Em 520(20)

5 10 / z- Tecan Ultra Evolution GENios Pro Safire Tecan Ultra Evolution Safire 4 40 2 Tecan Ultra Evolution Tecan Safire GENios Pro S-B/B GRE 485/535 COS 485/535 GRE 465/515 COS 465/515 GRE 485/515 COS 485/515 GRE 485/520 COS 485/520 2 25000 Greiner GRE Costar COS Tecan Ultra Evolution Ex 485(20)/Em 515(20) nm 3 25000 / 25000 / Greiner Costar Ex 485(20)/Em 515(20) 10 / z- Tecan Ultra Evolution GENios Pro Safire Tecan Ultra Evolution Safire 40 3 4 (S-B)/B (S-B)/B 1 1 [][]LGMSMB)/B-(SB)

6 /B(SRatio = S GFP RFU B RFU SM LGM Ultra Evolution Safire GENios Pro 3 Greiner 96 (S-B)/B Ratio Ratio Ultra Evolution Safire GENios Pro 4 S-B /B Costar Greiner Costar Greiner Coster Szabo-Scandic, Austria, Cat. Greiner Cat. No. 655090 96 TC GFP 25000 / 25000 / Ex 485(20)/Em 515(20) nm 10 / 40 4 LOD Greiner Costar Tecan Ultra Evolution Safire GENios Pro 10 / 40 z- Ultra 5 Evolution Ex 485(20)/Em 515(20) nm Ex 485(20)/Em 535(25) nm 5 eGEP- Tecan Ultra Evolution GFP 5 2 Ultra Evolution Safire GENios Pro(S-B))

7 /B COSGRE 4 Coaster Greiner Ultra Evolution Safire GENios Pro (S-B)/B 2 B(25000)Stdev*3*B)(F25000 LOD = LOD F 25000 RFU B RFU Stev 485/515 nm 485/535 nm Ultra Evolution 185 303 Safire 175 216 GENios Pro 245 439 5 Ultra Evolution Safire GENios Pro 2 GFP Costar LOD 6 50000 GFP 193 Hoechst 6 eGFP LOD 10 100 1000 10000 100000 10 1001000 10000100000 / RFU 485/515 nm 485/535 nm 101001000100001000001001000 10000100000 / RFU 485/515 nm 485/535 nm 5 GFP- Tecan Ultra

8 Evolution Greiner 2 / 6 a eGFP b c Costar Greiner d Tecan Ultra Evolution Safire GENios Pro eGFP 2 Tecan Safire Ex 492 nm Em 510 nm 10 15% 1 mrpw 3 1 1 mrpw eGFP 4 : 1 Ex 485(20)/Em 535(25), 2 Ex 465(35)/Em 515(20), 3 Ex 485(20)/Em 515(20), and 4 Ex 485(20)/Em 520(20) 2 Greiner 2 Costar 3 Ex 485 nm/Em 515 Greiner Costar (S-B)/B Tecan Safire (S-B)/B Tecan Ultra Evolution GENios Pro Safire 12 nm Tecan Ultra Evolution GENios Pro 20 25 nm Safire 2 485 nm 515 nm Tecan Ultra Evolution Safire (S-B)/B 4 GENios Pro (S-B)

9 /B 20% GFP- 6 GFP 192 / 50000 3 GFP Hoechst Tecan GENios Pro Safire Ultra Evolution z- Ultra Evolution Safire GENios Pro Ultra Evolution z- Costar ; Szabo-Scandic, Austria, Cat. No.: 3603 Greiner Cat. No.: 655090, 96 ,TC 2 4 GFP- 5 1 50000 eGFP LOD 5 Greiner 96 Ex 485/Em 515 nm Ultra Evolution z- 10 40 Ultra Evolution Safire 50000 190 2 Tecan GENios Pro LOD 250 Tecan Ultra Tecan Safire Ultra Evolution z- Safire Ex 485/Em 535 nm 2 1 5 Barbara Krammer University of Salzburg.

10 Institute of Physics and Biophysics Kristjan Plaetzer Tobias Kiesslich Christian Benno Monika Huber University of Salzburg, Institute of Physics and Biophysics Literature (1) Yang, F., Moss, L. G and G. N. Phillips: The Molecular Structure of Green Fluorescent Protein. Nat. Biotechnol. 1996, Oct, 14 (10), 1246-51. (2) Cubitt, A. B., Woollenweber, L. A. and R. Heim: Understanding Structure-Function Relationships in the Aequorea victoria Green Fluorescent Protein. Methods in Cell Biology, Vol. 58 Green Fluorescent Protein, Academic Press, 1999: 19-29. (3) Kain, S. R.: Enhanced Variants of the Green Fluorescent Protein for Greater Sensitivity, Diff