From 2012.igem.org
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| - | == Procedure ==
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| - | # Zentrifugation of the cell suspension at 6500 rpm at 4°C for 30 min.
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| - | # Resuspend the Pellet in 100 mL [[PBS buffer]] pH 7.4
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| - | # Transferration of the cell suspension into the celldisrupter and squash three times at 2.7 kbar
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| - | # Zentrifugation at 13.000 rpm at 4°C for an hour
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| - | # Load the prepared [[IMAC column]] with the hole supernatant and collect the flowthrough
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| - | # Wash the column two times with 20 mM imidazole in [[PBS buffer]] pH 7.4 and collect the floqthrough
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| - | # Wash the column in five steps with increasing imidazole concentrations (40mM, 60mM, 100mM, 200mM and 500mM) to elute the protein and collect the flowthrough
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| - | # Load the collected flowthrough on a 12% SDS-PAGE (Schägger)
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| - | # Combine the collected passages of the right protein and concentrate it to an amont of 5 mL
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| - | # To get rid of waste and undesired proteins perform a gelfiltration with FPLC (fast protein liquid chromatography)
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| - | # Usage of HiLoad 16/60 Superdex 75g from GE Healthcare
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| - | # Equilibration of the column with PBS buffer pH 7.4
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| - | # Loading 5 mL of the protein solution
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| - | # Elute the protein with pure PBS buffer pH 7.4 and collect the samples
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| - | # measure the protein concentration
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| - | # aliquot in 1 mL stocks and freeze in liquid nitrogen
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| - | # store at -80°C
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Latest revision as of 14:05, 9 September 2012
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