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| - | {{SUSTC-Shenzhen/removeStyles}}
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| - | {{SUSTC-Shenzhen/themeCss}}
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| - | {{:Team:SUSTC-Shenzhen/templates/nav-template}}
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| - | {{:Team:SUSTC-Shenzhen/templates/page-header|
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| - | title=Notebook|
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| - | subtitle=Element of an endeavor}}
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| - | {{SUSTC-Shenzhen/main-content-begin}}
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| - | {{:Team:SUSTC-Shenzhen/templates/notebook-main|
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| - | name=A-B Toxin|
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| - | date=2014|
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| - | goal=--to purify the protein}}
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| - | =Purification of the chimeric fusion protein via Ni2+ affinity chromatography=
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| - | ==Materials:==
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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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| - |
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| - | ==Protocol:==
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| - | 1. Resuspend the bead s and add 15ml ddH2O to per-wash
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| - | 2. Add 15ml binding buffer to balance the column and flow out all the binding buffer
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| - | 3. Close the flow valve , add 15ml supernatant to the column, incubate for 30min, stir the beads when the beads precipitate.
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| - | 4. Wash with 5ml binding buffer
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| - | 5. Wash with 10ml wash buffer
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| - | 6. Close the flow valve, add 1ml elution buffer, stir by pipetting, incubate for 2min, collect the eluent
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| - | 7. Repeat step 6 three times
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| - | 8. Wash with 15ml cleasing buffer
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| - | 9. Wash with 20ml 22% ethanol
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| - | 10. Store in 3ml 22% ethanol
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