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1. Buffer Sgeo Tactin Superflow 1 Triton X 100 1 Tween 0 3 CHAPS 2 Igepal CA 630 1 M NaCl Anti FLAGP M2 Agarose from mouse 5 Triton X 100 5 Tween20 0 1 CHAPS 0 1 lgepal CA 630 1 M NaCl transmembrane dgomain 50kDa 70kDa 50kDa2. 5 C 8 szep tag5 G8 strep F 5 GGGAATTCGGTACCGGATCCGGTGGAGGTGGAGGTGGAGGTGGATGGAGCCATCCGCAGTTT GAAAAGTAATCTAGAGC 3 G8 strep R 5 GCTCTAGATTACTTTTCAAACTGCGGATGGCTCCATCCACCTCCACCTCCACCTCCACCGGAT CCGGTACCGAATTCCO 3 coRI 2a pTD1 coRL X2al FLAG tag 3 2007 10 Copyright 2007 Shimadzu Corp All Rights Reserved SHIMADZ
3. 20 50 kL desthiobiotin Sgep Tactin Superflow 5 10 FLAGS_ tag 50 mM Tris HCI pH8 0 150 mM NaCI Buffer B PD 10 GE 17 0851 01 Anti FLAG M2 Agarose from mouse SIGMA A2220 50 1 1 mL Buffer B 5 mL PD 10 1 0 mL Buffer B 5 100 g mL FLAG Peptide SIGMA F3290 Buffer B 2 5 mL 20 50 gL 2
4. EcoRV lt gt 4 2007 10 Copyright 2007 Shimadzu Corp All Rights Reserved
5. BIOTECH Transdirect insect cel G8 FLAG F 5 GGGAATTCGGTACCGGATCCGGTGGAGGTGGAGGTGGAGGTGGAGACTACAAGGATGACGAT GACAAGTAATCTAGAGC 3 G8 FLAG R 5 GCTCTAGATTACTTGTCATCGTCATCCTTGTAGTCTCCACCTCCACCTCCACCTCCACCGGATC CGGTACCGAATTCCO 3 pTD1 coRI X2gl pTD1 G8 strep TAATACGACTCACTATAG MM T7 Promoter 5 Polyhedrin Leader Sequence GATATCGAATTCGGTACCGGATCCIGGTGGAGGTGGAGGTGGAGGTGGAITGGAGCOCATCCGCAGTTTGAAAAG coRV coRI Kpnl BamHl Gly X 8 spacer strep TAATCTAGA L_ stop Xba pTD1 G8 FLAG a ec ici T7 Promoter 5 Polyhedrin Leader Sequence GATATCGAATTCGGTACCGGATCCIGGTGGAGGTGGAGGTGGAGGTGGAIGACTACAAGGATGACGATGACAAG EcoRV EcoRI Ao gaz Gly x 8 spacer FLAG TAATCTAGA stop Xbal N C vector C EcoRI Ao BamHI
6. 2007 10 Copyright 2007 Shimadzu Corp All Rights Reserved SHIMADZ BIOTECH 5 Transdirect insect ce Anti FLAGS M2 Agarose from mouse 5 10 Transdirect secr ce7 1 mL 10 20 g N C 4 QO md A
7. FLAG tag C N 5 ATGNNNNNNNNNNNNNNN 3 FLAGR tag C 5 GGGGTACC TTACTTGTCATCGTCATCCTTGTAGTCINNNNNNNNNN 3 AI stop 1 2007 10 Copyright 2007 Shimadzu Corp All Rights Reserved SHIMADZ BIOTECH is Transdirect insect cel PCR DNA pTD1 DNA 2 mRNA mRNA RNA 100uxL mRNA Transdirect nsect ce 9 10 100wL 500 g mRNA 3 Transdirect nsect ce 1 mL mRNA 320 g 4 mM Methionine 20uL Reaction Buffer 300uL Insect Cell Extract 500uL H O to 10
8. 00 uu L 25 C 4 5 15 000 rpm 15 3 mRNA Nick V columns GE Helthcare sep tagP Strep Tactin Superflow QIAGEN 30001 30003 50 1 1 mL mRNA Nick columns bed volume 0 5 mL 50 mM Tris HCI pH8 0 300 mM NaCI Buffer A 5 mL 0 5 mL Buffer A 5 2 5 mM desthiobiotin SIGMA D1411 1G Buffer A 1 5 mL
9. SHIMADZ BIOT EC H i EE Transdirect insect cel C N sep tag IBA GmbH FLAG tag Sigma Aldrich Corp 1 cDNA pTD1 sep tagS N s eo tag5 N 5 ATGITGGAGCCATCCGCAGTTTGAAAAGINNNNNNNNNNNNNNN 3 C 5 GGGGTACC TTANNNNNNNNNN 3 kp stop FLAGS tag N FLAG R tag N 5 ATGGACTACAAGGATGACGATGACAAGNNNNNNNNNNNNNNN 3 C 5 GGGGTACC TTANNNNNNNNNN 3 Ao stop spep tagE C N 5 ATGNNNNNNNNNNNNNNN 3 s ep tag5 C 5 GGGGTACC TTACTTTTCAAACTGCGGATGGCTCCAINNNNNNNNNN 3 Ao stop
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