取扱説明書 (Version 1.0) 研究用試薬 CLD03, CLD07
Contents
1. CloverDirect GFP 1 100 GFP CloverDirect N 1 Ee mU CSET 2 1 CloverDirect Cl22. 7 Q amp A O RTS100 A O A 15 CloverDirect ATTO 633 tRNA Reagent for Site Directed Protein Labeling 8 E 1 1 MF20 nttD www olympus co jp Jp lisg genome m
3. 14 CloverDirect ATTO 633 tRNA Reagent for Site Directed Protein Labeling 1 2 SDS PAGE 3 1 2
4. UAG ProX tag E 1 2 a ATHA GSSG 10 mM mIkE NEHA 1 2
5. RTS100 E coli HY Kit UAG DNA BERK DNA mRNA E tRNA 80C tRNA 80 E tRNA 10 80C 2 tRNA E tRNA RIL RNase DNase E tRNA
6. Figure 6 1 0 251 IL SDS PAGE HiLyteF luor488 TAMRA 2UAG ProX tag 2UAG ProX tag CN f c N c c N c c N c c lt Sf E S lt E x S o S o S oO S o o 8 NS 2 de o V3 amp N Sa o Mas Ta 2 co Tae Cas Sa Q Cas 2 zeut a amp 2 gt e wo amp cn co ae Oo uw SL I ec Oo olL my ff ou c Ss o I xv c c S of xX cC c gt CG c Aa LL ao OL EF 90 6 LL au vor PS gt i a mm uu eal ne a P A ATTO633 2UAG ProX tag a EE 5 Figure 6 x gt S x gt z gt so E 8 3 8 8 2UAG AUG UAG a u amp o gt Uw ce o S 5 g
7. CloverDirect ATTO 633 tRNA Reagent for Site Directed Protein Labeling RTS100 E coli HY Kit 10 uL tRNA 1 uL X 10 uL 50 uL tRNA Chloramphenicol Acetyltransferase CAT page 10 Figu
8. 100 N 20 N 2 20 phenylalanine XHiLyteFluor 488 ATTO633 Figure 1 tRNA mRNA Gl oe N tRNA UAG UAG Figure 2 CloverDirect ATTO 633 tRNA Reagent for Site Directed Protein Labeling UAG UAG
9. ProX amp tag AUG UCU AAA CAA AUC GAA GUA AAC UAG UCU AAU GAG 3 Met Ser Lys Gln Ile Glu Val Asn Xaa Ser Asn Glu Figure 3 ProX amp tag Casel N ProX tag Case2 UAG UAG Prox tag UAG CloverDirect ATTO 633 tRNA Reagent for Site Directed Protein Labeling
10. 3 N N UAG UAG N 20 20 REE 4 ProX amp tag Prox tag Figure 3 CloverDirect N N i ii 2
11. FRET 1 2 E E E SDS PAGE TR JL ARDD ND EI SDS PAGE 16 CloverDirect ATTO 633 tRNA Reagent for Site Directed Protein Labeling 1 FRET analysis of protein conformational change through position specific incorporation of fluorescent amino acids Daisuke Kajihara Ryoji Abe Issei lijima Chie Komiyama Masahiko Sisido Takahiro Hohsaka Nature Methods 3 923 929 2006 2 47 2 124 128 2007 10 ant 7 S R tRNA an ae HiLyte Fluor 488 tRNA 497 525 CLDO1 CLDO5 TAMRA tRNA 546 575 CLDO2 CLDO6 ATTO 633 tRNA 629 657 CLDO3 CLDO7 Biotin tRNA CLDO4 CLDO8 ll URL http www proteinexpress co jp T260 0856 1 8 15 TEL 043 202 5755 FAX 043 202 5756 E mail tech
12. E UAG mRNA 2 E UAG UAA UGA lt i IRU TCESL E C His tag tag 1 2 N 20 3 N N 20 FLAG tag 4 ProX tag UCU AAA CAA AUC GAA GUA AAC UAG UCU AAU GAG Ser Lys Gin lle Glu Val Asn Xaa Ser Asn Glu Figure 4 UAG CloverDirect ATTO 633 tRNA Reagent for Site Directed Protein Labeling 5 1 E
13. 70 100 xg 1 3 Step7 2 mL Step8 Elute buffer 200 uL 10 70 100xg 1 Step9 Step8 Step10 Step8 9 X NaCl imidazole 13 6 CloverDirect ATTO 633 tRNA Reagent for Site Directed Protein Labeling E 1 a 2 DNase RNase HET CAD 80 2
14. CLDO3 CLDO y Ce ProteinExpress CloverDirect ATTO 633 tRNA Reagent for Site Directed Protein Labeling Version 1 0 mA ICIS MERIC SMW RG E3 2 C5USGtEA DC RTS100 E coli HY kit URL http www proteinexpress co jp 260 0856 1 8 15 TEL 043 202 5755 FAX 043 202 5756 E mail tech proteinexpress co jp CloverDirect ATTO 633 tRNA Reagent for Site Directed Protein Labeling CloverDirect tRNA Reagent for Site Directed Protein Labeling
15. tRNA mam J 7BR tRNA nm nm HiLyte Fluor 488 tRNA 497 525 CLDO1 CLDO5 TAMRA tRNA 546 575 CLDO2 CLD06 ATTO 633 tRNA 629 657 CLDO3 CLDO7 CloverDirect ATTO 633 tRNA Reagent for Site Directed Protein Labeling 3 E 1 UAG N 2 20 UAG tRNA Figure 1 tRNA UAG Figure 2 UAG
16. VAG 1 N page 11 Figure 6 2 VAG UAG N 20 20
17. proteinexpress co jp 17 CloverDirect ATTO 633 tRNA Reagent for Site Directed Protein Labeling 18
18. uL E coli 12 uL Step2 30 C 30 2 tRNA 80 C 2 AT Step4 SDS PAGE page 11 5 2 0 5 1 1 5 2 2 5 10uL TAMRA tRNA uL Figure 5 TAMRA tRNA TAMRA tRNA CloverDirect TAMRA Cat No CLDO2 CLD06 CAT SDS PAGE FIOR 10uL TAMRA tRNA 1uL 1009 10 CloverDirect ATTO 633 tRNA Reagent for Site Directed Protein Labeling SDS PAGE
19. 5 E SHY lt T ProX tag Prox tag 0 25 uL HiLyteFluor488 488nm 520 nm TAMRA 532nm 580 nm a ATTO633 635nm 670 nm a His tag i oo d sam oa ll CloverDirect ATTO 633 tRNA Reagent for Site Directed Protein Labeling His tag His tag GE His SpinTrap Column m His SpinTrap Column GE Wash buffer 20 mM Phosphate buffer
20. N 1 1 FRET 3 CloverDirect Bm tRNA 2 1 tRNA buffer RTS100 E coli HY kit ZE E 80 C E tRNA buffer 80 C 2 2 1
21. pH7 4 0 5 M NaCl 60 mM imidazole 0 1 Polyoxyethylene 23 Lauryl Ether Elute buffer 20 mM Phosphate buffer DH7 4 0 5 M NaCl 0 5 M imidazole 0 1 Polyoxyethylene 23 Lauryl Ether E SDS PAGE 12 CloverDirect ATTO 633 tRNA Reagent for Site Directed Protein Labeling Stepl His SpinTrap Column 2 mL 1 5 mL 1 Step2 70 100xg 1 Step3 500 uL Wash buffer 70 100xg 1 Step4 50 uL Wash buffer 350 uL Step5 10 70 100xg 1 Step6 500 uL Wash buffer
22. re 5 RTS100 E coli HY kit UL RNase 5 DNA 100 ng pL DNA 100 ng uL 5 mRNA 8 ug uL 12 1 10 tRNA 5 E coli 12 Total 0 tRNA tRNA RNase tRNA buffer CloverDirect ATTO 633 tRNA Reagent for Site Directed Protein Labeling Step1 tRNA 30 uL tRNA buffer tRNA tRNA buffer Step2 RNase DNA DNA mRNA Step3 Step1 tRNA
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