Finding the protein interacting with his-tagged Orf1 in M.smegmatis Orf1 Constructing Yeast two hybrid vector of EF-tu Preparing of Nothern blotting (confirming the expression of EF-Tu in 7H9+0.2%glucose or 7H9 30% CO ) Orf2 Transfomation pMsm orf2 vector in BL21 ->Induction using by IPTG (Isopropyl-β-DThioga lactopyranoside) ->Setting the induction condition ->purification of His-tagged Orf2 ->Confirming the his-tagged Orf2 using Western blotting ->Co-immuno-precipitation of the protein inter acting with His-tagged Orf2 from M. smegmatis Re-extract peptide interacting with Orf2 pMsmOrf2 5436 bp bla lacI His-tagged Orf2 orf2 pMsmOrf2-T/NcoI and XbaI Nco I (341) Xba I (1087)
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Finding the protein interacting with his-tagged Orf1 in M.smegmatis
Finding the protein interacting with his-tagged Orf1 in M.smegmatis. Orf1 Constructing Yeast two hybrid vector of EF-tu Preparing of Nothern blotting (confirming the expression of EF-Tu in 7H9+0.2%glucose or 7H9 30% CO ). Orf2 Transfomation pMsm orf2 vector in BL21 - PowerPoint PPT Presentation
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Finding the protein interacting with his-tagged Orf1 in M.smegmatis Orf1Constructing Yeast two hybrid vector of EF-tuPreparing of Nothern blotting(confirming the expression of EF-Tu in 7H9+0.2%glucose or 7H9 30% CO )
Orf2Transfomation pMsm orf2 vector in BL21->Induction using by IPTG (Isopropyl-β-DThiogalactopyranoside) ->Setting the induction condition->purification of His-tagged Orf2->Confirming the his-tagged Orf2 using Western blotting ->Co-immuno-precipitation of the protein interacting with His-tagged Orf2 from M. smegmatis Re-extract peptide interacting with Orf2
pMsmOrf25436 bp
bla
lacI His-tagged Orf2
orf2
pMsmOrf2-T/NcoI and XbaI
Nco I (341)
Xba I (1087)
Yeast two hybrid vector of EF-Tu
pGAD GH-EF-Tu9188 bp
Ampicillin resistance gene
EF-Tu
LEU2
Col E1 plasmid replication origin
S. cerevisiae ADH1 terminator
Eco RI (854)
Nco I (1968)
Sma I (844)
Xma I (842)
Bam HI (836)
Bam HI (1465)
Cla I (2052)
Cla I (3783)
Hin dIII (401)
Hin dIII (2402)
Pst I (852)
Pst I (4632)
Pst I (8123)
Apa LI (2215)Apa LI (5190)
Apa LI (6437)
Apa LI (6934)
Apa LI (8733)
Ava I (435)
Ava I (842)
Ava I (1397)
Ava I (1898)
Ava I (2066)
Ava I (7812)
pAS2-1 EF-Tu9567 bp
TRP1 coding sequence
GAL4 binding domain polypeptide
Ampicillin resistance gene
CYH2 gene
EF-Tu
Saccharomyces cerevisiae ADH1 promoter
2¥ì origin of replication
f1 single-strand DNA origin
pUC origin of plasmid replication
CYC1 terminator
Bam HI (6599)
Eco RI (5988)
Pst I (7189)
Nco I (5977)
Nco I (7102)
Apa LI (1590)
Apa LI (2595)
Apa LI (8072)
Apa LI (9318)
Hin dIII (2397)
Hin dIII (3284)
Hin dIII (5477)
Hin dIII (7399)
Ava I (294)
Ava I (3297)
Ava I (3305)
Ava I (5719)
Ava I (6531)
Ava I (7032)
B1 B2 B3 B4 M P1 P2 P3 P4
M:1Kb ladder
P: prey vector->1201bps
B: Bait vector->1201bps
Bait vector -> performing the sequencing
Prey vector ->performing the sequencing
Co-immuno-precipitation of the protein interacting with His-tagged Orf1 & Orf2 from M. smegmatis
174
117
76
51
39
26
Orf1 Orf2
174
117
76
51
39
26
IgGOrf1
Orf2
Purification of his-tagged Orf2 & Western blotting
M C E9 C E9M C FT W E1 E2 E5 E7 E9 E11
174117
7651
39
26
약 28KDa
MALDI-TOF analysis the protein interactiong with Orf1