取扱説明書 研究用試薬
Contents
1. 1 2 12 CloverDirect tRNA Reagent for Site Directed Protein Functionalization EX9 IRAY Avho k ERTER 1 2 SDS PAGE 3 12. 2 6 O amp A OQ RTS100 A
3. His SpinTrap Column GE Wash buffer 20 mM Phosphate buffer pH7 4 0 5 M NaCl 60 mM imidazole 0 1 Polyoxyethylene 23 Lauryl Ether Elute buffer 20 mM Phosphate buffer pH7 4 0 5 M NaCl 0 5 M imidazole 0 1 6 Polyoxyethylene 23 Lauryl Ether B SDS PAGE 10 Step1 Step2 Step3 Step4 Step5 Step6 Step Step8 Step9 Step10 CloverDirect tRNA Reagent for Site Directed Protein Functionalization His SpinTrap Column 2 mL 1 5 mL 1 7Z0 lt 100x 9 1 500 uL Wash buffer 70 lt 100x g T 1
4. Figure 4 1 0 25 1 uL SDS PAGE HiLyteFluor488 TAMRA ATTO633 2UAG 2UAG ProX tag ProX tag 2UAG ProX tag CN S S OM S S A S amp q S N c S ANA S sS JEdE JEHE JEJE 8 8 z 3 Qo 5 S Co 5 Q 5 S Ns sz NS 5 s NS 2 B ca 2 5 cog ag ca 2 5 cam 4 cass 9 cc a FCS goG opri iumoe9 gc ori umn9 Sm o gasorr umo9 sgoauorr tiumeo ox c lcoumrclIaxc SS c lco cITrIxcc SS SS SS Dr Oar SS Dr 0ALe zooogonduuwrnuwit rn Oar amp amp ooondumnurv E oooOnduir Figure 4 2UAG AUG UAG TM suh JI 2UAG ProX tag ProX tag ProX tag UAG o s q
5. TIRNA tRNAamber Xo aus aus mRNA 0 E BRI idis cai T UAG D tRNAamber UAG mRNA m f FORME Release factor UAG CGU AAU e UAG re Release factor 4 CCCG tRNA Go tRNAcccg SR TT d e A GCCC AGC CGGG CGU tRNAcccg CGGG mRNA PE 3 CCG 3 tRNA Qc muy CGG GUC UAA U o me UAA Stop CGG tRNA
6. TAG TAG 4 1 DNA FX ii AUC RZzJEBE tRNAS E colk ARBARA un j 16 en CloverDirect tRNA Reagent for Site Directed Protein Functionalization 10 URL http www proteinexpress co jp T260 0856 1 8 15 T EXEXRq A vav229MW TEL 045 202 5755 FAX 043 202 5756 E mail tech amp proteinexpress co jp 17
7. Figure 1 UAG CGGG CloverDirect tRNA Reagent for Site Directed Protein Functionalization B tRNA 5 tRNA buffer 5 X 1 300uL tRNA 80C 2 B 80 C B tRNA buffer 80C 2 B RTS100 E coli HY kit Roche Applied Science 3186156 UAG CGGG DNA DNA mRNA
8. Figure 1 UAA UAG UGA CGGG AGU ACC GUU GGC AAC GGU 5 GGC GGU CGGG AGU ACC GUU GGU AAC GGU CloverDirect tRNA Reagent for Site Directed Protein Functionalization 4 4 1 tRNA tRNA buffer RTS100 E coli HY Kit UAG CGGG DNA DNA mRNA B tRNA tRNA buffer
9. UAG a CGGG B TAMRA N N N ProX tag ProX tag Figure 2 Clover ecr ProX tag N ProX tag
10. 50 uL Wash buffer 350 yL 10 70 100x 2 1 500 uL Wash buffer 70 lt 100 x 9 1 3 2 mL Elute buffer 200 uL 10 70 lt 100x 2 1 Step8 Step8 9 NaCl imidazole 1 1 CloverDirect tRNA Reagent for Site Directed Protein Functionalization 5 B 1
11. o 15 CloverDirect tRNA Reagent for Site Directed Protein Functionalization 7 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 200060 2 Position specific incorporation of biotinylated non natural amino acids into a protein in a cell free translation system Takayoshi Watanabe Norihito Muranaka Issei Iijima and Takahiro Hohsaka Biochem Biophys Res Commun 361 794 799 2007 3 Comprehensive screening of amber suppressor tRNAs suitable for incorporation of non natural amino acids in a cell free translation system Hikaru Taira Yosuke Matsushita Kenji Kojima Kaori Shiraga Takahiro Hohsaka Biochem Biophys Res Commun 374 304 308 2008 4 Ffficient Incorporation of Nonnatural Amino Acids with Large Aromatic Groups into Streptavidin in In Vitro Protein Synthesizing Systems Takahiro Hohsaka Daisuke Kajihara Yuki Ashizuka Hiroshi Murakami and Masahiko Sisido J Am Chem Soc 121 34 40 1999 5 FW 72 3 247 253 2007 6
12. tRNA http www proteinexpress co jp j products reagent cloverdirect 1 htm 15 CloverDirect tRNA Reagent for Site Directed Protein Functionalization 9 tRNA tRNA 0 p HO0 P 0 f 0 0 ey T pdCpA Ho 0 cine ES tRNA Y HsN R m E HH i pdCpA n ii tRNA AUC 3 CA tRNA tRNA UAG CGGG coli LEF
13. DNase RNase Ne 2 Neu 3 Ne 4 TEIT TAS Ne D Neu 80 C UAG CGGG ProX tag B 1 2 3 Ne GSSG 10 mM
14. fij His SpinTrap kit GE 28 9321 71 Ultrafiltration Membrane ULTRAFREE 0 5 Centrifugal Filter Devices 10k UFV5BCOO X CloverDirect tRNA Reagent for Site Directed Protein Functionalization 3 UAG CGGG R AE TSN B N 20 http www proteinexpress co jp j products reagent cloverdirect 7 htm B mRNA 2
15. tRNA 30 uL tRNA buffer B tRNNA 80 tRNA 80 tRNNA B tRNA RNase DNase B tRNA CloverDirect tRNA Reagent for Site Directed Protein Functionalization
16. RTS100 E coli HY Kit 10 uL IS2XIUC tRNA 1 pL X 10 ruL 50 uL X tRNA Chloramphenicol Acetyltransferase CAT page 8 Figure 3 RTS100 E coli HY kit ul RNase 5 DNA 100 ng
17. C ProteinExpress sg CloverDirect tRNA Reagents for Site Directed Protein Functionalization Version 1 1 RTS100 E coli HY kit aape ceo p URL http www proteinexpress co jp T260 0856 1 8 15 TEL 043 202 5755 FAX 043 202 5756 E mail tech amp proteinexpress co jp CloverDirect tRNA Reagent for Site Directed Protein Functionalization 1 10 LO EE EA E E E 2 0
18. 0 25 uL F 2 3 S a OSEEE ESSESSESEE HiLyteFluor488 488nm 520 nm sw E TAMRA 532nm 580 nm erit cud uen ATTO633 635nm 670 nm cp w fi His tag X ProX tag CloverDirect 24 6 477 V2 8 003 E 73 32 CloverDirect tRNA Reagent for Site Directed Protein Functionalization 4 3 His tag His tag His tag GE His SpinTrap Column
19. N ProX tag UAG D AUG UCU AAA CAA AUC GAA GUA AAC n UCU AAU GAG 3 Met Ser Lys Gln Ile Glu Val Asn Xaa Ser Asn Glu Figure 2 Prox tag CloverDirect tRNA Reagent for Site Directed Protein Functionalization Casel N ProX tag Case2 UAG CGGG
20. 3 ARETO MT 4 pis X M 6 4 1 RR 6 4 2 ee 9 4 3 His tag rc 10 ME 12 QQA WM 13 p 14 SU EU RE 15 RR 16 9 EK 5 EE 17 CloverDirect tRNA Reagent for Site Directed Protein Functionalization 1 tRNA Reagents for Site Directed Protein Functionalization EE 1 UAG CGGG 4 tRNA UAG CGGG TOR UAG Arg tRNA CGGG 100
21. 30 0 30 2 tRNA 80 Step4 SDS PAGE page 9 4 2 Lm eT g O Wt J O O 0 5 1 1 5 2 2 5 3 10LL TAMRA tRNA uL Figure 3 TAMRA tRNA TAMRA tRNA CloverDirect TAMRA Cat No CLD02 CLD06 Chloramphenicol Acetyltransferase CAT TAMRA SDS PAGE 10uL TAMRA tRNA 1uL 100 CloverDirect tRNA Reagent for Site Directed Protein Functionalization 4 2 SDS PAGE
22. ProX tag WB C His tag 47128 His tag B AG C UAG UAA UGA B CGGG CGG tRNA CGG CGG CGU CGC B CGGG
23. uL DNA 100 ng uL 5 mRNA 8 ug uL 17 1 10 tRNA D E coi 12 Total SU tRNA tRNA RNase tRNA buffer CloverDirect tRNA Reagent for Site Directed Protein Functionalization Stepl tRNA tRNA buffer tRNA 30 ul tRNA buffer Step2 RNase DNA DNA mRNA Step3 Step1 tRNA 5 uL E coli 24 4z F 12 uL Step2
24. AiR 47 2 124 128 2007 14 CloverDirect tRNA Reagent for Site Directed Protein Functionalization 8 for Fluorescence Labeling CloverDirect CR110 X AF tRNA 5 CR110 X Abs Em 498 521nm CloverDirect HiLyte Fluor 488 AF tRNA iryte Fluor 488 Abs Em 497 525nml CloverDirect TAMRA X AF tRNA 5 6 TAMRA X Abs Em 546 575nml CloverDirect ATTO 633 AF tRNA IATTO633 Abs Em 629 657nml CloverDirect ATTO 655 X AF tRNA ATTO655 X Abs Em 633 684nm for Fluorescence Labeling Clover Direct Biotin AF tRNA Biotin Clover Direct Biotin X AF tRNA Biotin X CloverDirect Biotin XX AF tRNA Biotin XX for Post translational Modification CloverDirect Tyr PO H tRNA O phospho Tyrl CloverDirect Lys Me tRNA methyl Lys CloverDirect Lys Me tRNA dimethyl Lys CloverDirect Lys Ac tRNA acetyl Lys for Unnatural Mutagenesis PEG CloverDirect PEG4 AF tRNA Methyl PEG4 CloverDirect PEG8 AF tRNA Methyl PEG8 CloverDirect PEG12 AF tRNA Methyl PEG12 CloverDirect BPA tRNA p benzoyl phenylalanine CloverDirect AcPhe tRNA p acetyl phenylalanine CloverDirect 2M aZzoAla tRNA p phenylazophenyl alanine
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