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| - | {{SUSTC-Shenzhen/main-content-begin}}
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| - | name=A-B Toxin|
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| - | date= |
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| - | goal=to purify the protein}}
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| - | =8.23-8.26=
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| - | ==preliminary purification of chimeric fusion protein: GD5 and TEG==
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| - | ===Materials:===
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| - | Plasmid pSW55-GD5(chimeric fusion protein carries a yeast transcription factor Gal4,an antibody fragment specific for the tumor-associated ErbB2 antigen and an internal DT translocation domain)
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| - | Plasmid pWF47-TEG(chimeric fusion protein carries a yeast transcription factor Gal4, an EGF receptor ligand TGF-a, and an internal Pseudomonas exotoxin A translocation domain)
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| - | DH5-a,
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| - | BL21,
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| - | Amp LB plates,
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| - | 100ug/ml ampicillin LB liquid media,
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| - | TIANprep Rapid Mini Plasmid Kit(TIANGEN○R),
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| - | 100ug/ml ampicillin LB liquid media containing 0.6% glucose,
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| - | Lysis buffer: 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10 µM ZnCl2, 0.3 mM PMSF, 8 M urea
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| - | Binding buffer: 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10 µM ZnCl2, 0.3 mM PMSF, 8 M urea, 10 mM imidazole
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| - | Wash buffer: 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10 µM ZnCl2, 0.3 mM PMSF, 8 M urea, 50mM imidazole
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| - | Elution buffer: 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10 µM ZnCl2, 0.3 mM PMSF, 8 M urea, 250 mM imidazole
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| - | Cleansing buffer: 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10 µM ZnCl2, 0.3 mM PMSF, 8 M urea, 1 M imidazole
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| - | Ni2+ affinity chromatography
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| - | ===Methods===
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| - | ====8.23====
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| - | 1.We got the plasmids of GD5 and TEG from Winfried Wels, Institute for Experimental Cancer Research, Tumor Biology Center, Breisacher Strasse 117, D-79106 Freiburg, Germany
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| - | 2.Transform these two plasmids into DH5-a, and incubated at Amp LB plates over night at 37oC.
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| - | ====8.24====
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| - | 1.Pick up a single colony of TEG and of GD5
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| - | 2.Transform these two colonies into 100 µg/ml ampicillin LB liquid media, grow 12h at 37ºC, 200rpm
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| - | 3.Extract plasmids GD5 and TEG by TIANprep Rapid Mini Plasmid Kit(TIANGEN○R)
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| - | 4.Transform plasmids GD5 and TEG to BL21, grow overnight at Amp LB plates at 37ºC
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| - |
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| - | ====8.25====
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| - | 1. Pick up single colony of plasmids GD5 and TEG to 2 ml LB medium containing 100 µg/ml ampicillin and 0.6%glucose and grow 2.5h at 37ºC and 200rpm (11:15am)
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| - | 2. Dilute the culture to 100ml l fresh in LB medium containing 100 µg/ml ampicillin and 0.6 % glucose , grow at 37ºC to an OD600 of 0.6 (2:00pm)
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| - | 3. TEG OD600=0.30, GD5 OD600=0.38 (3:50pm)
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| - | 4. TEG OD600=0.38, GD5 OD600=0.51 (5:00pm)
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| - | 5. TEG OD600=0.55, GD5 OD600=0.64 (6:00pm)
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| - | 6.Add IPTG to a final concentration of 0.5mM and expression is induced for 1.75h at 37ºC, 200rpm (6:10pm)
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| - | 7. The cells were divided into 6 centrifuge tubes( 7:55pm)
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| - | 8. Harvest the cell at 4ºC by centrifugation at 9000rpm for 15 min. (8:30pm)
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| - | 9. Resuspend the cell in 27ml lysis buffer (1g cells = 25ml lysis buffer)
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| - | 10. Thel cells are lysed by sonication for 3 min on ice (5s on, 3s off) (10:10pm)
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| - | 11. The lysate is gently shaken for 1.5 h at room temperature (26ºC)
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| - | ====8.26====
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| - | 12. Centrifuged the cells at 4ºC in for 40 min at 9200rpm and then collect the supernatant (8.27 0:40am)
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| - | 13. Repeat step 12 (1:40am)
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| - | 14. The supernatant is collected, 10 mM imidazole final concentration is added and stored at 4ºC. (2:00am)
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| - | 15. Purify GD5 and TEG via Ni2+ affinity chromatography by the protocol Purification of the chimeric fusion protein via Ni2+ affinity chromatography
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| - | 16. Proteins GD5 and TEG are determined by SDS-PAGE and
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| - | Coomassie brilliant blue staining
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| - | ===Results:===
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| - | ====Supposed Results:====
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| - | We can see only one band in the elution samples of TEG and GD5, TEG is 38kDa, GD5 is 68kDa
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| - | ====Actual Results:====
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| - | {{SUSTC-Image|wiki/images/a/ad/SUSTC-Shenzhen-plate1.JPG|TEG bacteira}}
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| - | Schematic representation of the TEG fusion gene in the E.
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| - | coli expression plasmid pWF47-TEG
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| - | {{SUSTC-Image|wiki/images/3/36/SUSTC-Shenzhen-plate2.JPG|GD5 bacteria}}
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| - | E.coli which expressed plasmid pSW55-GD5
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