Team:SUSTC-Shenzhen/Notebook/A-B Toxin/Purify
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===Materials:=== | ===Materials:=== | ||
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) | 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) | ||
+ | |||
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) | 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) | ||
+ | |||
DH5-a, | DH5-a, | ||
+ | |||
BL21, | BL21, | ||
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Amp LB plates, | Amp LB plates, | ||
+ | |||
100ug/ml ampicillin LB liquid media, | 100ug/ml ampicillin LB liquid media, | ||
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TIANprep Rapid Mini Plasmid Kit(TIANGEN○R), | TIANprep Rapid Mini Plasmid Kit(TIANGEN○R), | ||
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100ug/ml ampicillin LB liquid media containing 0.6% glucose, | 100ug/ml ampicillin LB liquid media containing 0.6% glucose, | ||
+ | |||
Lysis buffer: 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10 µM ZnCl2, 0.3 mM PMSF, 8 M urea | 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 | 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 | 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 | 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 | 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 | Ni2+ affinity chromatography | ||
Revision as of 05:48, 12 October 2014
Notebook
Element of an endeavor
Contents |
A-B Toxin
2014 --to purify the protein8.24-8.28
preliminary purification of chimeric fusion protein: GD5 and TEG
Materials:
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)
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)
DH5-a,
BL21,
Amp LB plates,
100ug/ml ampicillin LB liquid media,
TIANprep Rapid Mini Plasmid Kit(TIANGEN○R),
100ug/ml ampicillin LB liquid media containing 0.6% glucose,
Lysis buffer: 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10 µM ZnCl2, 0.3 mM PMSF, 8 M urea
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
Wash buffer: 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10 µM ZnCl2, 0.3 mM PMSF, 8 M urea, 50mM imidazole
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
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
Ni2+ affinity chromatography
Methods
8.24
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
2. Transform these two plasmids into DH5-a, and incubated at Amp LB plates over night at 37oC.
8.25
1. Pick up a single colony of TEG and of GD5
2. Transform these two colonies into 100 µg/ml ampicillin LB liquid media, grow 12h at 37oC, 200rpm 3. Extract plasmids GD5 and TEG by TIANprep Rapid Mini Plasmid Kit(TIANGEN○R)
4. Transform plasmids GD5 and TEG to BL21, grow overnight at Amp LB plates at 37oC
8.26
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 37C and 200rpm (11:15am)
2. Dilute the culture to 100ml l fresh in LB medium containing 100 µg/ml ampicillin and 0.6 % glucose , grow at 37C to an OD600 of 0.6 (2:00pm)
3. TEG OD600=0.30, GD5 OD600=0.38 (3:50pm)
4. TEG OD600=0.38, GD5 OD600=0.51 (5:00pm)
5. TEG OD600=0.55, GD5 OD600=0.64 (6:00pm)
6.Add IPTG to a final concentration of 0.5mM and expression is induced for 1.75h at 37C, 200rpm (6:10pm)
7. The cells were divided into 6 centrifuge tubes( 7:55pm)
8. Harvest the cell at 4C by centrifugation at 9000rpm for 15 min. (8:30pm)
9. Resuspend the cell in 27ml lysis buffer (1g cells = 25ml lysis buffer)
10. Thel cells are lysed by sonication for 3 min on ice (5s on, 3s off) (10:10pm)
11. The lysate is gently shaken for 1.5 h at room temperature (26oC)
8.27
12. Centrifuged the cells at 4 °C in for 40 min at 9200rpm and then collect the supernatant (8.27 0:40am)
13. Repeat step 12 (1:40am)
14. The supernatant is collected, 10 mM imidazole final concentration is added and stored at 4°C. (2:00am)
15. Purify GD5 and TEG via Ni2+ affinity chromatography by the protocol Purification of the chimeric fusion protein via Ni2+ affinity chromatography
16. Proteins GD5 and TEG are determined by SDS-PAGE and Coomassie brilliant blue staining
Results:
Supposed Results:
We can see only one band in the elution samples of TEG and GD5, TEG is 38kDa, GD5 is 68kDa
Actual Results:
Schematic representation of the TEG fusion gene in the E. coli expression plasmid pWF47-TEG